Genetics Exam 4

Each bacterial colony is a clone, carrying a specific cloned DNA sequence

the whole thing plated on the medium is a colony, and each are a clone of some specific DNA sequence

How to use a retro virus for gene therapy

-use retrovirus to deliver new gene -the bacterium containing the plasmid with the cloned normal gene > the target gene is inserted into a genetically disabled retrovirus -the retrovirus infects target cells that you want to insert your target gene into and transfers the target gene into it -the infected cells are then cultured to ensure the target gene is active -and then re-implanted back into person -useful because they insert DNA into chromosome

Some Current Applications of Plant Biotechnology

2011 data: 90% of corn, soybean, and cotton crops are GMO -the method described above is used to make these crops

When using DNA fingerprinting to determine the paternity of a child, how many of the child's 13 str loci should match the father's?

6 or 7 -probably because half of your DNA is from dad, and half from mom, so half should match (at each loci, you have an allele from mom, and an allele from dad)

Plants are being "engineered" to be more resistant to insects or viruses, to be herbicide tolerant

75% of soybeans and cotton in the US are herbicide resistant

Different Restriction Enzymes cut different DNA sequences

Bacteria that make a restriction enzyme also make a corresponding methylase. This corresponding methylase stops the bacteria from cutting its own DNA at sites that the restriction enzyme would otherwise recognize in the bacteria.

What genes cause cancer and why?

Oncogenic (cancer-causing ) Viruses carry oncogenes -DNA tumor viruses: Papilloma, Epstein Bar, Hepatitis B: Viral genes activate DNA replication in host -RNA tumor viruses - retroviruses: Infection by virus causes mis-expression of host genes -in humans RNA tumor viruses aren't a primary cause of cancer, because they don't directly cause cancer, but they may lead to mutations that do so cancer is a secondary affect of the virus -organisms other than human do have retroviruses that are the direct cause of cancer when the virus enters the cell and expresses its genes, it causes mis-expression of host genes

PCR Uses DNA Polymerase to copy DNA in the test tube repeated rounds of replication

Specificity of PCR depends on choosing the right primers and the annealing Temp used

The most abundant sequences in the human genome are

TRANSPOSABLE ELEMENTS - more transposable elements and repeats occur in more complex organisms

Glo Fish and Glo Pigs!

engineered to express the gene for green fluorescent protein cloned from jelly fish

Eukaryotic Genomes Vary Greatly in Size

the bigger the genome, the more repeated sequences there are

Genome

the complete set of DNA in a single cell of an organism

Genomics

the study of genomes

Eukaryotic genomes

-linear, genes less densely packed, -monocistronic with introns in the genes -amount of repeat DNA and intron size increase with evolutionary complexity -Amount of noncoding spacer DNA and the number and size of introns increases with evolutionary complexity in eukaryotes -the average gene has 12 introns in it -Duchenne Muscular Dystrophy gene -- 2 Mb, >70 introns! ***biggest gene

You can do Locus-specific PCR of a VNTR or a VNTR southern blot to analyze someones DNA profile (VNTR with a southern blot requires a bigger sample of DNA)

***Colin Pitchfork convicted in 1987 of rape and murder based on DNA evidence

Knockout Mice: Targeted DNA Integration Via recombination In Embryonic Stem Cells

*Don't need to be worry about this but be aware -direct DNA into specific sites in mice and only in mice -done by homologous recombination -Create plasmid w seqeunces that are homologous to sites in chromosome and look where the DNA entered chromosome through double crossover * produce a plasmid that has sequences homologous to a target site in the chromosome* -used to turn genes off by making a frameshift which inactivates that particular gene -put plasmid growing in cells in tissue culture that s in an antibiotic medium > this shuts down native gene because only the cells containing the plasmid are antibiotic resistant this is called a KNOCKOUT -you can do this w eukaryotes in tissue culture but you can't get animals back from it but only in mice - use embryonic stem cells and grow them in tissue culture and put it into an already grown embryo and these stem cells will participate in making a embryo -used for studies for models for human disease like muscular dystrophy and cystic fibrosis

Because of genetically engineered foods...

*we've had genetically modified crops approved for human consumption since 1990, improvements in agriculture and yields has plateaued probably -Crop yields have more than quadrupled in the last century (developed countries) -Half of the increase has been due to improvements in agricultural practices, half to plant breeding -In the future genetic engineering is going to play an Increasingly important role

Genetic and Fossil Evidence Suggests Modern Humans Migrated out of Africa in Two Waves

-70,000 years ago to India, Southern Asia and Australia -50,000 years ago to Europe and Northern Asia -Europe and Asia were already occupied -- Neanderthals

Applications of Gene cloning: Recombinant DNA

-Biotechnology: the genetic alteration of animals, plants, and bacteria **Transgenic organisms: In eukaryotes the introduced DNA must insert into a chromosome to be retained and expressed.

Bacillus thuringiensis - BT toxin Insect Resistance

-Bt is a naturally occurring soil bacterium that produces a protein that is toxic to insects -BT has been used as an insecticide for over 100 years, without evidence of environmental problems or human or animal toxicity -Organic farmers use BT dust for insect control *BT causes the digestive tract of insects that ingest it to lyse -effective against caterpillar/mosquitoes/beetles - used in agriculture for over 100 years -Comes in the form of powder which is a dry bacterial culture -Genetically modified plants that makes BT makes its usage easier

How to make transgenic animals

-Transfection - cloned DNA Introduced into tissue culture cells -Add target DNA to cell culture in pea tree dish -DNA enters nucleus and is expressed but is not integrated into chromosome -1 colony forms/10^6 cells treated with 40 pg gene DNA *DNA must integrate into a chromosome to be maintained and stably expressed *The site of DNA integration is essentially random *DNA integration may cause a mutation *****Site where new DNA is integrated occurs via nonhomologous DNA repair mechanism, but it puts this piece of DNA anywhere, which is a problem that was only solved in mice

Making transgenic mice

-Transgenic Mice by injection of DNA into a fertilized egg -then the fertilized eggs are placed in the oviduct of a receptive (previously pregnant) female -RECOMBINANT DNA CAN BE PUT INTO A SPECIFIC SPOT IN MICE

Overproduction of the Gene for Growth Hormone in Fish

-contain transgenic growth hormone genes -transgenic atlantic salmon have copies of chinook salmon growth hormone next to a constitutive expressed (unregulated, constantly active) promoter -no adverse health effects from added gene

Retroviruses can acquire host genes

-first you have a nonacute retrovirus - the genome of the virus can infect the cell but not transform it -the retrovirus can then pick up a copy of the host proto-oncogene during its infection of the cell and integrate it into its genome -the cellular proto-oncogene may be mutated during the process of transfer into the virus, or it may be expressed at abnormal levels because it is now under the control of viral promoters -these new retroviruses can infect and transform normal cells into tumor cells, they are ACUTE TRANSFORMING RETROVIRUSES *the transfer of gene from host cell into the viral genome allows the virus to infect and transform cells MAKING IT AN ONCOGENIC RETROVIRUS -An acute retrovirus has an additional part - these are the genes that cause cancer, somehow the virus picked up a normal gene while infecting the host cell, which mutates normal gene and causes cancer -This can happen because the virus planks down into chromosome and gene is right next door, the neighboring piece of DNA can end up in the retrovirus during its replication

In 2009 the FDA Approved transgenic Goats for the production Of anticoagulant

-put human anticoagulant gene next to promoter for beta caesin, a common protein in milk, they were able to target anticoagulant expression in the mammary gland, so this is highly expressed in the milk, and then isolate it from the milk

Which of the following disorders would be the easiest to treat by gene therapy and which would be the hardest? 1. Muscular dystrophy 2. Cystic fibrosis 3. Hemophilia

ANSWER: Easiest 3, Hardest 1 easiest to target the circulatory system that is why hemophilia is the answer -harder to target muscles

Cancer is infectious, can be transmitted from person to person

ANSWER: disagree -cancer is not infectious and cannot be transmitted from person to person - one type of cancer that is transmissible is wiping out the Tasmanian devil - if you get cancer cells from someone else your immune system will kill it off, if you have a defective immune system it may be transmissible

Most cancers are caused by viruses

ANSWER: disagree there are some cancers that are caused by viruses, some viruses directly cause cancer, but most cancer doesn't have anything to do with virus

There are some inherited predispositions to cancer

Breast Cancer - genes affected are BRCA1, BRCA2 - recombination repair Hereditary nonpolyposis colon cancer - due to mismatch repair XP (xeroderma pigmentosa) - nucleotide excision repair -if you have the mutated gene you are at a greater risk for developing that cancer -Many of these genes affected are involved in DNA repair, causing the mutations and cancer WHICH ties mutation to cancer

Analysis of cloned genes - DNA sequencing

Dideoxynucleotide sequencing - has an -H not an -OH at the 3' end so it terminates replication - no where to form phosphodiester bond to the next nucleotide - YOU NEED .... -DNA polymerase -DNA template -primer -dATP, dGTP, dCTP, dTTP -small amount of a didNTP that is labeled 1. primer is annealed to DNA template 2. Then reaction mixture with DNA poly, all the bases, and the ddNTP (tagged with fluorescent dyes, each base has a diff color that this is attached to AKA ddATP - pink and ddGTP is green etc) 3. Eventually there will be a ddNTP inserted at every location so that each strand differs in length by 1 nucleotide because during primer extension it sometimes puts in ddNTP instead of dNTP 4. the products of this are added to a single lane on a capillary gel electrophoresis, and the bands are read by a detector system 5. the sequence is found by the extension of the primer, and is read from new strand not template strand

DNA from an organism is cut by a restriction enzyme and so is the vector DNA, both cut by the same enzyme, and then they can base pair with their complementary tails, and then gaps are sealed with DNA ligase THIS MAKES RECOMBINANT DNA

Done in a test tube at low temperature, this is necessary for hydrogen bonds to stabilize and stick together. Ligase seals the phosphodiester bonds and now they're bonded from two different sources

Law and Regulation of GMOs

FDA/EPA/USDA - GM crops must be shown (by the manufacturer) to be "substantially equivalent" to nonGM versions of the crop. Toxicity and allergenicity of the crop are assessed based on the nature of the protein product of the genes that have been inserted. If they are deemed safe US law does not require labeling

in 1990, Young survivor of ADA deficiency: treated at age 4 with transformed T-cells -in 2000, 20 babies are Successfully treated For SCIDX but 3 of them get lymphoma

First done in 1990, and she got a correction, she got 4 or 5 treatments, and she got 25% of her immune system back, it is enough that she will survive -in 2000, 20 babies treated for SCID X they did 2 bone marrow replacements on these kids, and they had 95% success with this, most kids got 50% of their immune system back, then 3/20 of he kids got cancer, and 2 were treated and 1 died, and in these kids this virus put DNA where regulation of cells was important thus causing cancer -in 2002 human gene therapy stopped to get around this problem

Which of the following sequences is a palindrome and could be a restriction enzyme recognition site? >GAATTC> <CTTAAG<

GAATTC

Cloning and sequencing of genes is providing unexpected information on how genes function and evolve

Human Genome Project: Human - "finished" 2003 Drosophila -- done 1999 Caenorhabditis (worm) -- done 1997 Arabidopsis (plant) -- completed 2000 Yeast -- done 1996 E. coli -- done 1995 Mouse -- 2004 *Sequenced more than just humans - these different genomes are cross informative - more info about human genome by looking at other organisms than just humans alone -everything has been sequenced at this point - all in genbank

Criticisms of GMOs

Safety -- Food Allergens Consumer choice - Labeling Bio Pollution Patents and control of food supply US decided that these crops must be substantially equivalent - have to show toxicity of protein product of the gene you put in (protein that dna codes for) -test to see if these proteins bring with them issues of toxicity or Allergenicity - that you can directly test -Bio pollution - biological polltuion - can these plants hybridize with the genes of native plants and then this can spread **For Example - with corn and cotton, there are no wild plants for the hybrid to cross with so there are no genes for it to hybridize with > worried that it can get away into wild plants -issue of patents and food supply - big companies that are producing seeds farmers are using > they have control over food supply

Using STRs to follow human history

The Y chromosome does not undergo recombination, so related males inherit identical STR alleles - haplotypes -Y chromosome is directly inherited from father to son - less genetic variability in Y chromosome than autosomal chromosomes (not sex) -even 2 unrelated males may have the same Y profile if they share a distant ancestor

Southern Blot of DNA from 4 Species: Human, mouse, hamster, and cow -Cut with EcoRI or HindIII or PstI -Probed with a fragment of the human cystic fibrosis gene

This is called a zoo block - they use animal DNA and probed it with the human cystic fibrosis gene to see if it would hybridize - aka is their DNA complementary to our DNA sequence for cystic fibrosis gene? -The cow showed some hybridization to the human gene, yet not in the same places as the human -The mouse and hamster don't have any hybridization - showing a bigger evolution between us and hamsters/mouse than us and cows -When you have a piece of DNA from 1 species and hybridize it with another species the sequences aren't the same, so hybridization isn't the same

Human insulin (humulin) was the first human protein to be produced in bacteria using recombinant DNA

To make recombinant human insulin... -mature insulin in made of 2 polypeptide chains: A (21 amino acids) and B (60 amino acids) -synthetic genes to make A and B were made by oligonucleotide synthesis (63 nucleotides for A and 90 for B) -These oligonucleotide sequences are inserted into separate vectors at the tail end of a cloned E. coli lacZ gene -The recombinant plasmids were transformed into E. coli host cells, where the B-gal/insulin fusion protein was synthesized and accumulated in cells -B-gal/insulin fusion proteins were extracted from host cells and purified -insulin chains were released from B-gal by treating with cyanogen bromide -insulin subunits were purified and mixed to produce a functional insulin molecule

Prenatal Testing: Amniocentesis ASO or FISH can be used for genetic diseases

Use amniotic fluid and obtain the fluid and the fetal cells from it - culture this on a pea tree dish and analyze it using recombinant DNA methods for looking at genetic and biochemical analysis -you can do it before insemination if you couple it with in vitro fertilization

Tissue culture allow us to grow entire plants from fragments of stems, roots or leaves

You can genetically modify plants in tissue culture > make genetic modification of cells and take these cells to make a plant -make pieces of plant by manipulating culture conditions > you make roots and put them in soil and they'll grow **Easiest to make genetic modification is in tissue culture

Mutations in the EGF (epidermal growth factor) and neu growth factor RECEPTORS result in stimulation of cell division even in the absence of the hormone

when it loses the functional growth factor receptors, it causes it to not need the hormone to bind to cause cell division, so it is always active -some mutations cause it to bind the hormone and dimerize it doubling the output of the cell division -mutation of the neu growth factor receptor is a base substitution -mutation in EGF receptor is a deletion

Genetic Modification Of Plants Using Agrobacterium tumefaciens Crown Gall Disease

-Agrobacterium carries a plasmid and transfers a piece of that plasmid into plant cells upon infection (transfer T-DNA) -the T-DNA contains genes that stimulate auxin and cytokinin synthesis in infected plant tissues -Best way of introduction of new DNA uses natural soil bacteria that is a cellular overgrowth - tumor induced plasmid and transfers it into plant chromosome and the piece of DNA that goes into plant cell contains genes that messes up plant hormone production causing the mass of cells -people found that you can take out whatever DNA you want and insert it into the Tumor induced plasmid (Ti) which will become part of the original plant chromosome

Retroviral Oncogenes are altered versions of normal cellular genes

-Altered activity or over expression of oncogenes results in cancer (gain of function mutations) -Only one allele of a proto-oncogene needs to be mutated or misexpressed in order to trigger uncontrolled growth SO oncogenes are a dominant cancer phenotype *viral oncogene is an oncogene in a virus

Gene Therapy for ADA/SCID

-Bacterium carrying plasmid with cloned normal human ADA gene and a genetically disabled retrovirus are coupled so the cloned ADA gene is incorporated into the virus -bone marrow cells are removed and cultured from SCID patient, and the engineered retrovirus is added to cell cultures -the retrovirus infects blood cells, transfers ADA gene to cells -genetically modified bone marrow cells are bulked up and returned to patient and see if you get a therapeutic response

Herbicide Resistance --- Glyphosate and others

-Broad spectrum herbicide -Blocks EPSP synthase, an enzyme in the pathway for synthesis of aromatic amino acids. -Animals lack this enzyme, so toxicity of glyphosate is low -Breaks down quickly in soil (1-2 weeks) -Cheap -Transgenic plants expressing an EPSP synthase gene from bacteria are resistant to glyphosate -Herbicide resistance - grow plants without chemicals is not realistic to support our whole population - spraying with organically synthesized compounds is always a risk, this particular compound is the least toxic we know of but not completely nontoxic, this kills all plants by blocking EPSP which blocks amino acid synthesis (all animals get amino acids from diet) glyphosate has low toxicity to all animals and it degrades quickly in soil -FDA put this on the carcinogen list SO it has the potential to cause cancer but it is not known to cause cancer, only people at risk are people who make or use this compound -need to spray less with a more toxic herbicide because it persists longer in the soil

Amplification of MYC and MDM2 in Neuroblastoma cells

-Cancer cells have genomic instability -increase in chromosome number -amplification of specific bits of the chromosome -in this picture the 2 cancer genes are amplified as small DNA fragments that remain separate from the chromosomal DNA within the nucleus -MYC is red and MDM2 is green -Normal chromosomes are blue -With MYC, you have pieces of DNA that have gotten off the chromosome and replicated in left -in the right there is amplification on the chromosome increasing the protein product from these cancer causing cells

Carcinogens cause cancer by causing mutations

-Cancer is a genetic disease - what we know is that the association between cancer and mutations is tight ALL OF THE FOLLOWING ARE CARCINOGENS THAT CAUSE CANCER: -Radiation -Polycyclic hydrocarbons -benzopyrene -Nitrosamines -Aflatoxin

Cancer cells exhibit: *loss of growth control - cells continue to divide *altered cell appearance - partially dedifferentiated *ability to move to new site - metastasis

-Cancer is due to accumulation of mutations in somatic cells of genes involved in growth control -Cancer is a diverse set of diseases, many types -general features of all cancers: loss of growth control so cells continue to divide when they shouldn't, there is altered cellular appearance because they're expressing a different set of genes which alters their phenotype so they look different, cells lose their identity and move to different sites in the body, usually they stay in the same specific area (metastasis) We know that cancer is due to the accumulation of genes in growth control MITOSIS

Chapter 16 -- Cancer

-Cancer results from interaction of genes and environment -Today, the most frequent is skin, then lung -if you looked at the same list of cancer frequencies from the1900s, the list would be the same except lung cancer would be number 10 instead of 2

Evidence that Cancer is a Genetic Disease

-Carcinogens are mutagens -Predisposition to cancer is inherited in some families: Mutations in genes encoding DNA repair enzymes result in predisposition to cancer -Specific chromosome rearrangements are seen in some cancers: CML and the "Philadelphia chromosome" -Cancer cells show genomic instability: Translocations, deletions, gene amplifications *Cancer causing chemicals are mutagenic - you can measure how frequently chemicals cause damage to DNA by measuring the rate at which you treat salmonella with the chemical and measure how much mutations you get -predisposition to cancer can be inherited, in particular the enzymes for DNA repair promotes cancer -in some cancers you have very specific chromosome rearrangements which suggests that typical genetic rearrangements are causing cancer -cancer cells show wide scale genomic instability: insertions, deletions, amplifications, translocations - creating abnormal cells with weird genetic expression

Philadelphia Chromosome - Chronic Myelogenous Leukemia

-Caused by a reciprocal translocation between chromosome 9 and 22 - placing BCR and ABL gene next to each other, now cell division regulator ABL is under control of promoter of BCR and then you'll have over production of ABL which kicks off the cancer -presence of this translocation causes the cancer 95% of the time -IT IS SPECIFICALLY THE TRANSLOCATED 22 CHROMOSOME WITH A SMALL PIECE OF CHROMOSOME 9 ON THE BOTTOM OF IT (9 has the ABL gene)

Process of analyzing DNA with STRs using PCR

-Each primer set is tagged with 1 fluorescent dyes - red, blue, green, yellow -Each primer is designed to amplify DNA fragments from specific loci, so different loci can have the same color -primers are specific for flanking the STR locus and labeled with blue dye -the double stranded DNA s denatured, primers annealed, and each allele (repeat sequence) is amplified by PCR in the presence of dNTPS and Taq DNA polymerase > then separated by cap electro -the sizes of amplified DNA fragments from one color can help differentiate the loci products that have the same color -after amplification, the sizes of amplified fragments are measured by capillary electrophoresis - uses a capillary tube with gel to run a current through it, smaller fragments travel further -a laser detects the fluorescent fragments through the tube -after this the information is translated using computer system finding the size and quantities of each fragment -this DNA profile is then compared to that of other peoples

Transgenic Organisms

-Genetic modification of bacteria, animals, and plants -Introduction of genes from other organisms -clone gene from donor organism into a plasmid -modify gene sequence to permit expression in target organism -introduce plasmid into cells of target organism

The difficulties with human gene therapy primarily have to do with delivery of the DNA to sufficient numbers of target cells in the patient to get a therapeutic response, generally less than 10% of treated cells

-MLV - only infects dividing cells, tends to integrate in transcriptionally active regions - cancer prone -Lentiviruses (HIV) - infect both dividing and nondividing cells, less likely to integrate in transcriptionally active regions -Adenovirus and AAV - DNA genomes, do not integrate into the chromosomes but will infect nondividing and dividing cells -Viruses can trigger immune responses in the patient particularly if the patient has been exposed to the virus before (adenovirus particularly bad in this way)

Microsatellites (STRs/ short tandem repeats) are used for DNA Fingerprinting

-Microsatellites - 2 to 7 base sequences repeated less than 50 times -Different individuals can differ in the number of copies at a given site. -"STRs" - short tandem repeats -known locus STR D8S1179 is made up of a 4-base pair sequence, TCTA, repeated 7-20 times depending on the allele (number of copies of STR)

Most oncogenes are involved in the hormone signal pathways leading to expression of genes that stimulate cell division

-Oncogenes are genes whose normal function is to promote cell division (under appropriate conditions) -Most oncogenes are genes that code for proteins that code for hormone response -all cell division is regulated by these hormone responses that promote or inhibit cell division -Ras proteins transmit signals from the cell membrane to the nucleus, stimulating the cell to divide in response to growth factors -Ras protein switches off from active (GTP) and inactive form when growth factors aren't binded (GDP) -growth factors bind to the growth factor receptors on the cell membrane, activating Ras -the active GTP bound form of Ras then sends its signals through cascades of protein phosphorylations in the cytoplasm -the end point of these cascades activates activate nuclear transcription factors that stimulate expression of genes whose products start the cell into the cell cycle -once Ras sends its signal to nucleus, it transforms active GTP to GDP, to inactive it -mutations of proto-oncogene Ras into an oncogene will prevent it from turning GTP into GDP, so the cell thinks there is always a growth factor present and always divides **mutations in any of the Ras signaling pathway phosphorylations make them stimulated all the time in the absence of Ras, making them potential Oncogenes

Virus Resistance: transgenic plants expressing a piece of the viral genome are often resistant to the virus

-Particularly effective with RNA viruses and the mechanism of resistance involves RNA interference ***Papayas and Squash -In the 1990's Hawaiian Papaya production dropped by more than 50% due to Papaya ringspot virus. -Resistant varieties began to be planted in 2000 and the Papaya industry has recovered -this was a success in papayas -RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of siRNA mRNA molecules. -siRNA comes from the binding of the virus with the RNA and then the dicer cuts it up and the RITS inactivates it -Use genetic modifications to build RNA response to a virus so the plant is ready for the virus before it comes - this is done with RNA interference

Human Gene Therapy - aims to transfer normal genes into a patients cells

-SCID -- ADA deficiency, SCIDX -The Bubble-boy died of SCID at age 11 Good Candidate Disorders for Gene Therapy Trials? *Simple recessive disorder due to a single gene *Life threatening with little or no other treatment available *Disorder of the circulatory system - SCID - people born w recessive disorders that eliminates their immune system -People with SCID are sensitive to any infection that comes their way -There are 2 different mutations than can cause SCID -This seemed like good candidate for gene therapy in humans - it is a simple recessive disorder for 1 gene (put in correct gene), the disorder is life threatening with little or no other treatment, and this is a disorder of the circulatory system - so you can take cells from the bone marrow and alter them and put them back -anything with the circulatory system should be fixable w gene therapy

Golden Rice - Increased Vitamin A content (carotenoid synthesis genes from daffodil)

-Soybeans and canola with monounsaturated Oleic acid instead of polyunsaturated oils -It is possible to change the nutritional value of things - like in rice. It is genetically modified to increase the expression of vitamin A content

2009 - successful correction of X-linked adrenoleukodystrophy (ALD) in two young boys using a lentivirus (retrovirus that can infect non-dividing cells)

-Thousands of clinical trials are underway to treat cystic fibrosis hemophilia Parkinsons HIV and more -there is not a cancer problem with this, the problem is DNA delivery

*****Genetic corrections of cells in the germ line are ruled out because they would be heritable putting future generations at risk.

-Treatment is targeted to specific organs in the patient -Disorders of the circulatory system are most amenable because bone marrow stem cells can be removed and grown in tissue culture **in the circulatory system you can take cells out, make genetic modifications to them in a dish, and then put the cells back > the biggest problem is to get DNA into the target organism in big enough amounts to fix the problem - delivery mechanism is tailored to the organism

Retrovirus Life Cycle

-Viral RNA enters host cell -DNA copy of Virus made -Viral DNA integrates into host chromosome -Viral RNA made by transcription -Release of new viral particles (PROTEINS)

Plants and Mammals are also used as "bioreactors" for production of human proteins because bacteria do not glycosylate proteins

-bioreactors - living factories - that will continuously make the product containing the desired therapeutic protein that can be isolated in a noninvasive way -regardless of the host, therapeutic proteins may then be purified from host cells

Transgenic plants are made using the soil bacterium Agrobacterium tumefaciens -bacterial EPSP synthase gene provides resistance to the broad spectrum herbicide Glyphosate

-herbicides kill plants by inhibiting the action of the enzyme EPSP sythase -to make herbicide resistant plants you ... -take the EPSP synthase gene from bacteria and fuse it to the right promoter and poly A signals CREATING a fusion gene -introduce fusion gene into TI plasmid that doesn't have tumor genes and then this is transformed into host plant cells in tissue culture -Plants expressing the EPSP synthase gene are grown from the tissue culture - you know they're expressing it if they are survive on glyphosphate medium *cells that acquire the EPSP synthase gene are able to synthesize large quantities of it, making them resistant to herbicide glyphosate -use a promoter that comes from a virus to obtain a high level of expression in all cells throughout the plant, use specific promoters if you want it to be specific placement like in leaves or stems, etc

Transgenic Animals -- mice, sheep, pigs, etc *Microinjection of DNA into a Fertilized Egg *Egg is implanted into uterus

-isolate newly fertilized eggs in host female animal -inject purified cloned DNA containing a vector and the transgene of interest into nucleus of egg -only a small amount of the transgenic eggs will contain the transgenic DNA into the egg cell genome -injected eggs are then placed into the oviduct of a animal previously impregnated by a male -once baby is born it is screened for the trans gene via PCR -the integrated DNA must be present in germ (gamete) cells so it is passed to all offspring -most F1 aren't homozygous for the transgene so the F1 siblings must mate to make homozygous transgenic animals

Retrovirus explained and their way of replacing their DNA with the therapeutic DNA

A retrovirus is any virus belonging to the viral family Retroviridae. All The genetic material in retroviruses is in the form of RNA molecules, while the genetic material of their hosts is in the form of DNA. When a retrovirus infects a host cell, it will introduce its RNA together with some enzymes into the cell. This RNA molecule from the retrovirus must produce a DNA copy from its RNA molecule before it can be considered part of the genetic material of the host cell. Retrovirus genomes commonly contain these three open reading frames that encode for proteins that can be found in the mature virus. Group-specific antigen (gag) codes for core and structural proteins of the virus, polymerase (pol) codes for reverse transcriptase, protease and integrase, and envelope (env) codes for the retroviral coat proteins (see figure 1). The process of producing a DNA copy from an RNA molecule is termed reverse transcription. It is carried out by one of the enzymes carried in the virus, called reverse transcriptase. After this DNA copy is produced and is free in the nucleus of the host cell, it must be incorporated into the genome of the host cell. That is, it must be inserted into the large DNA molecules in the cell (the chromosomes). This process is done by another enzyme carried in the virus called integrase (see figure 2). Now that the genetic material of the virus is incorporated and has become part of the genetic material of the host cell, we can say that the host cell is now modified to contain a new gene. If this host cell divides later, its descendants will all contain the new genes. Sometimes the genes of the retrovirus do not express their information immediately. Retroviral vectors are created by removal op the retroviral gag, pol, and env genes. These are replaced by the therapeutic gene. In order to produce vector particles a packaging cell is essential. Packaging cell lines provide all the viral proteins required for capsid production and the virion maturation of the vector. These packaging cell lines have been made so that they contain the gag, pol and env genes. Early packaging cell lines contained replication competent retroviral genomes and a single recombination event between this genome and the retroviral DNA vector could result in the production of a wild type virus. Following insertion of the desired gene into in the retroviral DNA vector, and maintainance of the proper packaging cell line, it is now a simple matter to prepare retroviral vectors (see figure 3). One of the problems of gene therapy using retroviruses is that the integrase enzyme can insert the genetic material of the virus in any arbitrary position in the genome of the host. If genetic material happens to be inserted in the middle of one of the original genes of the host cell, this gene will be disrupted (insertional mutagenesis). If the gene happens to be one regulating cell division, uncontrolled cell division (i.e., cancer) can occur. This problem has recently begun to be addressed by utilizing zinc finger nucleases or by including certain sequences such as the beta-globin locus control region to direct the site of integration to specific chromosomal sites.

DNA fingerprinting

A test to identify and evaluate the genetic information-called DNA in a person's cells

All cancer cells grow faster than normal cells

ANSWER: disagree there are some cancers that are fast growing and some that are slow growing, very diverse, prostate cancer is slow growing

Making transgenic humans is considered unethical because:

ANSWER: we cannot control where the recombinant DNA will go when it integrates into the chromosomes -You can't control where DNA goes when you add it in, so you create the possibility of mutations in genes that are important - not unethical to make genetic modifications to certain cells, BUT if you make bad mutations in transgenic organisms it can be passed on to the next generation WHICH IS unethical

The Neanderthal Genome has been sequenced

ancestors evolve into neanderthals 350,000-500,000 years ago - and they died out 30,000 years ago and modern homo sapiens take over -All non-African modern humans have 2-4% Neanderthal DNA sequences

Northern blotts are also used to study splicing reactions - like in this case ^, the splicing is done differently in the heart and the brain because they have different amount of RNA

tells us about alternative splicing, size of mrna transcript, and estimate relative transcriptional activity of a gene

proto-oncogenes encode transcription factors that stimulate expression of other genes, signal transduction molecules that stimulate cell division, and cell cycle regulators that move cells through the cycle

when a proto-oncogene is mutated or overly expressed and contributes to the development of cancer it is an ***ONCOGENE***

Limitations of PCR

-info about DNA is needed to make primers -minor contamination of DNA can cause problems for accuracy (ex: skin cells from researcher contaminate samples from crime scene) -Usually, DNA poly in PCR only extends primers for short distances, not to the end of the strand, SO PCR is usually used to amplify relatively short pieces of DNA

PCR (powerpoint notes)

-repeated rounds of steps -target DNA and excess of primers and DNA polymerase and the 4 bases, heat up to denature, and then cool the mixture to temp so the primers will just manage to base pair w target and polymerase will start to copy, then heat back up and put it back through the cycle again -doubling of target sequence

cDNA = "complementary DNA" made from mRNA

cDNA synthesis - uses Reverse Transcriptase to make a DNA copy of mRNA 1) mix mRNAs with oligo-dT primers (single stranded) and these bind to the poly A tail. oligo primer is (TTTTT) 2) reverse transcriptase extends the oligo-dT primer and makes a complementary DNA copy of the mRNA 4) The RNA part of the hybrid (because now you have one strand of DNA and 1 RNA) is partially digest by RNAse H, the 3 ends of remaining RNA serves as primers for DNA poly 1, to make the second strand of DNA 5) and the opposing strand of DNA is made with DNA poly 1 and ligase to make the final double stranded cDNA molecule

Two types of Gene Libraries

*Genomic DNA libraries -- cloned fragments of DNA isolated from chromosomes; represents all DNA sequences -used for cloning regulatory sequences (promoters), studying genome organization, sequencing of whole genomes, if you want to find introns *cDNA libraries -- cloned DNA copies of mRNA isolated from cells or tissues -used for isolating expressed genes -used to study genes expressed in certain cells and tissues under certain conditions

Analysis of cloned genes - Blotting - Use blotting to see how the gene is put together inside the cell or use blotting to measure expression

*Southern blotting is where DNA is cut with a restriction enzyme, run on a gel, and probed for a specific sequence, used to study structure of a gene or chromosome -used for which clones in a library contain a given DNA sequence *Northern blotting isolates RNA (takes specific mRNA from a certain type of cell/tissue, separated by gel electrophoresis, then transferred to a membrane to be probed) and then is run on a gel, and probed for a specific sequence - to study gene expression ***probes for the presence of mRNA complementary to a cloned gene

Two competing Groups Sequenced the Human Genome: Celera Inc. (Craig Venter) and International Human Genome Consortium (Francis Collins)

-"Draft" Sequence Published Feb 2001 -"Finished" Sequence 2003 -human genome project was an international project - worked collaboratively - Celera company helped in the late 1990's and sequenced faster and cheaper -these 2 groups were competing against each other, in 2000, clinton got them to negotiate and publish there data together at the same time in the same month ***Celera just wanted to identify as many genes as possible so they dropped out of the project, and human genome consortium finished the job

Cloning by PCR - Polymerase Chain Reaction

-Allows amplification of specific DNA sequences -Used to clone genes without making a library -Wide range of applications in genetics, molecular biology, evolution, medicine, criminal cases

Next Gen Sequencing the quest for the $1000 genome

-Current work focuses on sequencing different individuals to see how we differ and how these differences correlate with health and disease --- We are all 99.9% the same -The search for SNPs - single nucleotide polymorphisms - single base changes in the genome that usually cause disease -Sequencing has be revolutionized: cost came way down, and so this opens new avenues for new things, start looking at what the differences are between humans and how does this relate to disease -Look for snaps that correlate loss of functionality (SNP) - even at this cost it would take 30,000$ to sequence the genome 8 times to be accurate - only need to sequence coding part which you can do for less than 1000$

Southern blotting

-DNA can come from several sources -DNA samples are cut into fragments using 1 or more restriction enzymes, fragments are loaded onto agarose gel and separated by gel electrophoresis -The DNA in the gel is denatured with alkaline treatment to form single stranded fragments -Gel is then overlaid with DNA binding membrane (usually nylon) -To get the DNA fragments to the membrane by placing this on top of a wick (sponge) in buffer solution (gel is directly on top of sponge with membrane on top of it), with paper towels and weight on top of it - this draws buffer up through the gel, transferring the DNA fragments to the membrane -Membrane (filter) is placed in a heat sealed food bag with solution containing single stranded radioactive DNA probe; probe hybridizes with complementary seqeunces -Membrane is washed to removed unbound probe, dried, x-ray film is applied

Human Genome Consortium

-DNA from 8 males was sequenced - 23.1 billion bp sequenced -Human genome size - 3.2 Gb only 2.9 Gb was sequenced: centromeres and telomeres were skipped because they have very little DNA and are very repeated, they are difficult to sequence because of so many repeats so the areas around them are ignored -Gaps - about 5% of the sequence was in gaps between clones, Sequencing accuracy about 99.9% - "draft genome" -"Complete Sequence" 2003: 99.99% accuracy, some 300 gaps still remained -Human genome project doesn't say where the DNA came from - did it 8 fold coverage: didn't sequence females because they're missing the Y -In 2001 when the published they had 95% of the sequence, 5% had gaps, accuracy of 99.9% - this is draft sequence -2003 human genome project is done - they got it to 99.99% accuracy - about 300 gaps remaining, can't have it all but have most of it

Celera

-DNA from five subjects was selected for genomic DNA sequencing: two males and three females -one African-American, one Asian-Chinese, one Hispanic-Mexican, and two Caucasians -27.3 million sequence reads; 14.9 billion bp of sequence -when they published, Celera said they had DNA from 5 different people, different ethnic mix, human genome is only 3 billion base pairs but you have to sequence it multiple times to get an accurate reading with no gaps, sequenced each genome 5 times, which is barely enough to get a draft sequence

Genomic DNA Cut with a Restriction Enzyme Individual DNA Bands are not usually seen Because there are so many different fragments

-DNA has been cut with different restriction enzymes then run on different lanes, and the DNA has been stained w fluorescent dye, the right shows bands containing DNA sequences complementary to probe SHOWING hybridization -No bands on left because when you take DNA and cut w ECOR1 you expect to get a lot of fragments of different lengths, so the bands are in there but theres so many they appear as a smear ****SIZE OF A BAND SHOES THE SIZE OF A RESTRICTION SITE

You can also use Allele-Specific Oligonucleotides for genetic testing to find single nucleotide differences, but that are not limited to just the restriction enzyme cutting sites (like with RFLPs)

-DNA is extracted and the B globin gene is amplified using PCR, this is then denatured and spotted onto strips of DNA binding membrane and then probed with an ASO for either the normal B globin gene or mutant. If hybridized with the normal and they are homozygous dominant (normal allele, not sickle) then they will have the strongest hybridization, if heterozygous then they will have some hybridization, if homozygous recessive they will have no hybridization to the probe *only identical nucleotide sequences can hybridize to ASO -Way to detect single base pair substitution: using hybridization using specific short pieces of DNA -This is for sickle cell -Used to detect single base seqeunces so you can distinguish these 2 molecules binding to the DNA

Genetic Testing for Point Mutations

-Genetic detection of Sickle cell anemia -MstII RFLP -Use this to detect individuals that are heterozygous, homozygous recessive, or homozygous dominant for sickle cell anemia -Use the region where the point mutation can occur in the restriction site as a probe, for the first exon of the gene - if it is normal - you get 2 bands (GAG) mutant is (GTG) *This only happens if the single base change is right in the restrction enzyme site which is unlikely to be the case but it can happen

Chapter 19: Applications of Gene cloning Recombinant DNA

-Identification and Isolation of Important Genes in Humans and other Organisms -Screening for genetic disorders -DNA fingerprinting -- identification of individuals -Biotechnology -- genetic alteration of animals, plants, and bacteria -Gene Therapy -- correcting genetic disorders

RFLPs allow detection of inheritance of chromosome segments in families that are linked to a disease gene

-Inheritance of RFLPs is Codominant -RFLP allows u to detect the inheritance for genetic variance - imagine you have an RFLP that's inherited with association with an inherited disorder, take DNA from diff people cut with a restriction enzyme and hybridize with a probe > detect diff in where the enzyme cuts bc you'll have slightly different sequences in each -probe needs to be close or linked to gene you're seeing you might have, nearby will work too -Detecting genotype of people with respect to disorder is easy -Use southern blot to see the difference in fragments that are cut using restriction enzymes - DNA fragments are separated with gel electrophoresis, transferred to a nylon membrane, visualized with southern blot hybridization, and probed for mutation region

Gene Sequence Comparisons Help Identify Gene Function: Human NF1 and Yeast Ira Genes

-Ira and NF1 proteins both regulate the Ras protein -More and more genes are cloned and studied from different organisms, and knowing the functions of some of these genes in one organism can help predict the function of these genes in a different organism ***this is a comparison at the amino acid level with a human NF1 gene and the yeast ira gene, the NF1 gene was cloned years ago but no one knew what this protein did, when the yeast ira sequence was cloned, it turned out that it was similar to NF1 and ira gene has a known function: SO we now know about NF1 gene too -the yellow boxes are identical matches between the 2 genes, the lines are chemically similar but not identical -20-30% matching is actually significant, 5% is random similarities, you expect similarities because of a lot of mutations have occured since we evolved from this yeast ***organisms are used as model systems to be informative about the human genome

To explain ^

-Limitations in PCR: not specific in finding a sequence - primers are hard to design, make sure primers don't hit multiple sites in genome, the temperature where you anneal is really important -Make sure primers don't bind to off target sites and if they do, after 1 round of replication, it will bind perfectly to non perfect site -Anneal temperate based on MP of primers, run 3 degrees cooler than what you find it to be -You can amplify something unintended -enzymes can make errors: taq polymerase doesn't have proof reading function, will introduce errors into DNA that you're copying - when it puts in wrong base it stops replicating and reaction terminates, can't copy long pieces of DNA with taq polymerase bc of error rate - to get around this they do a long pcr with an enzyme w proof reading function

Cloning the gene for Huntington's Disease -- "Positional Cloning"

-Nancy Wexler cloned the Huntington's gene in 1991 -You look for linkage between inheritance of the disorder and DNA markers that have been mapped to specific sites on chromosomes -5000 related individuals in Venezula who were segregating for HD - including 100 who had the disease -FIRST SUCCESS -Nothing was known about Huntington's disease except that it was inherited - she looked for the disorder and inheritance of RFLPs, map it -connection between the dominant allele for a disease and how that RFLP in the dominant allele, recessive allele is cut differently and is not linked to the disease

Many important genes have been cloned using mapped RFLPs

-Neurofibromatosis -Muscular Dystrophy -Cystic Fibrosis -Huntingtons disease -Proof that the correct gene has been cloned?? -individuals with the disorder will have mutations in the candidate gene -Proof would be that individuals with the disorder have some sequence that people that don't have it don't have

GENETIC SCREENING

-Once a Gene for a disorder has been cloned the cloned gene can be used to identify individuals with the disorder using RFLPs *Detection of b-thalassemia - for the beta globin gene -Deletion or insertion mutations are easiest to detect In this version, there is a deletion that removes exon 3 > detected because there is an enzyme that cuts on either side of beta globin gene, and people with the mutation to cause sickle cell, like here, there band is shorter than with people with the normal allele, aka it'll be closer to the bottom -use pattern matching to detect mutant -All grey means you have 2 normal alleles -This method works well if mutant allele has big insertion or deletion in it, which is the case for this beta knot (mutant allele) -A single base change would have to be right in the restriction site to detect it

Analysis of cloned genes - restriction mapping

-Once you clone a gene you can make a map of it using restriction enzymes -Once you clone a gene you break it up into smaller pieces to take a closer look at it > and that is done with restriction maps -Clone DNA into a plasmid > cut plasmid with different restriction enzymes so you can make a restriction map and see where they are cut within the plasmid -If you get 2 size markers, that means the enzyme cut twice -The size markers indicate how far the cuts are from each other: EcoRI cuts twice at 7 and 3, so the distance between the 2 cuts in 1 direction will add up to 7, and the other add up to 3

To identify genes the computers look for ORFs (open reading frames) and other features of genes

-Once you have the sequence the job is finding the genes, you can use computers algorithms to find sequences consistent w genes, look for open reading frames with no stop codons -Open reading frames have no stop codons aka its probably exons, and identify exons this way, look for promoter sequences/splicing seqeunces -train computers to find this, and find the likely genes, this is annotating the genes, if an annotated gene maps back to an EST then you really have the right gene, verifies the annotation GOAL: FIND ALL THE GENES

Disadvantages of PCR

-PCR is not necessarily specific for the intended sequence -The primers and annealing temperature determine the specificity of PCR -Short versus Long PCR - PCR is not necessarily accurate -Taq Polymerase lacks 3'-5' exonuclease activity

PCR is a rapid method of DNA cloning > eliminates the use for host cells for cloning

-PCR used to amplify target DNA seqeunces that are initially present in small quantities -double stranded target DNA + DNA polymerase + and 4 DNA bases (A/T/C/G) are added to test tube -some info must be known about target DNA in order to synthesize 2 oligonucleotide primers (single stranded) -these primers bind to complementary DNA after they have been denatured, and are extended by DNA polymerase at the 3' end to make a clone -each cycle doubles the number of DNA molecules in the reaction -Specific DNA polymerase is used bc not all of them can handle the temperature change of annealing and cooling - Taq polymerase -DNA used can come from many samples - even used for forensics like hair samples and dried blood

DNA Fingerprinting -- Identification of Individuals

-RFLP differences between individuals -VNTRs - Variable Number Tandem Repeats "Minisatellites" short sequences (2-50 bases long) multiple copies at many different sites individuals vary in the number of copies 2-100 copies per site at ~ 40 sites -FE, if you examined 4 different VNTR loci, and each had 20 possible alleles, there would be 4^20 possible genotypes in this profile -someones unique DNA profile can be found for each person if 5-6 loci are analyzed - this is due to the large number of possible VNTRs and alleles -different alleles > from different repeat lengths

Restriction Enzymes

-Restriction endonucleases, cut DNA in a sequence specific manner -Recognition sites are Palindromic sequences: nucleotide sequence reads the same on both strands of the DNA when read in the 5' to 3' direction. -Each restriction enzyme recognizes a specific restriction site and cuts the DNA in a specific way **G/A cut: enzyme does this. Then theres a complementary overhanging ends called sticky ends, and these will be used for reassociation (different pieces from different sources) and they will stick to each other using complementary base pairing. -EcoRI have sticky ends, the vector must also been cut by the same restriction enzyme

New methods of DNA Sequencing are Driving down the cost and time required. The hope is that we will soon be able to sequence individual genomes for $1000

-Revolutions in DNA sequencing - ways to make DNA sequencing cheaper and faster > goal is to bring the cost of sequencing genomes down -Human genome sequencing itself took 10 years and took 3 or 5 billion dollars > now it can be done in a matter of days -You can target specific spots in genome and get a lot of info into medical background

Several Types of "Vectors" are used in cloning, they differ in the size of the "inserts" they can carry

-Standard Plasmids -- inserts < 10 kbp -Bacteriophage Vectors -- inserts 10-50 kbp -BAC Vectors -- inserts 100 kbp, ***All to know- different vectors are used to carry different size pieces of dna, so this makes them used for different purposes. -standard bacterial plasmids can hold about 10,000 bases of dna -Viruses-lambda- can hold a piece of DNA up to about 50,000 base pairs, typical size 25,000. this is a virus where its lysogenic, enters bacterial cell and becomes dormant. So to use bacteriophage as a vector, what has been done is to isolate the pieces at the end and you don't need the genes in the middle. Use these ends to clone DNA into it and the virus will package it. The f plasmid- only part is needed is the part for replication. ***lysogeny - viral DNA is integrated into bacterial chromosome, so every time the chromosome is replicated the viral DNA is replicated and passed down

Exome Sequencing - Personalized DNA Sequencing

-Take DNA from an individual and hybridize it to a gene chip containing sequences that match all human exons (protein coding genes). -After hybridization, recover the exons from the slide and sequence them. -Cost ~ $1,000 (1% of the genome but all the genes) -Hybridize DNA onto a slide and where u have spotted oligonucleotides that correspond to every exon, wash the slide to wash away anything that different hybridize, then wash it so the exons come off, take the exons and sequence it -Used to screen tumors to find an effective treatment

RFLPs Can be Mapped - Mapped RFLPs can be used to map and then clone genetic disorders

-Take a 3 point cross from 2 different people and you have different probes and fragment sizes and you can look to see if there is linkage between different alleles with these RFLPs -anything that says D1 is a RFLP site mapped to chromosome 1 - once they're mapped you can look at inheritance of RFLP and alleles of genes

Cloning DNA yields a "gene library" - collection of clones that contains all the DNA sequences of an organisms genome

-The "library" is a collection of clones 1. Isolated DNA from the organism 2. Cut DNA with Restriction Enzymes 3. Insert DNA fragments into a "Vector" (plasmid) 4. Introduce recombinant DNA into host cell E. coli) 5. Each bacterial colony contains a single recombinant DNA clone *restriction enzyme used for whole genomes is YAC Major challenge: how to identify the specific clone that has the gene you want? Screening Libraries

Predictions based on annotation and analysis of protein functional domains and motifs: genes in the human genome that have been assigned on the basis of similarity to proteins of known function (in other organisms) (slide 11 set 31)

-Use computers to predict what the unknown genes do using blast to compare predicted (unknown) gene to everything else that has ever been cloned that has info for what that gene does - if 2 genes are similar according to blast, they have proteins with similar function -Doesn't tell you which genes it codes for only tells us that it codes for a specific transcription factor, not which ones -molecular function unknown: 40% unknown, we don't know what is expressed and coded for by these genes

DNA is cut with a restriction enzyme that cuts outside of the VNTR

-VNTRs have codominant inheritance - so you'll see the bands for each allele at each loci they're testing -take a piece of DNA, cut it with a restriction enzyme that cleaves on either side of the VNTR repeat region -the digested DNA is separated by gel electrophoresis and then run on a southern blot -briefly, separated DNA is transferred from the gel to a membrane and hybridized with a radioactive probe that recognizes sequences within the VNTR region -expose membrane to x-ray film, the pattern of bands is measured, with larger VNTR repeat alleles remaining at the top of the gel, and smaller VNTRs which migrate more rapidly, being closer to the bottom ***THE LENGTH OF THE VNTR REPEATS REFLECTS A DIFFERENT ALLELE

Expression of b-globin genes during development

-different members of the beta gene family have slightly different functions, and are active in their own time in development -People with sickle cell > treat it to improve expression of delta because it will make a normal beta chain and increase the good beta in people with sick cell disease

slide 4 set 32 ????

-figured out

GWAS - Genome Wide Association Studies -The search for genes responsible for complex traits using SNPs FOR EXAMPLE - Type II diabetes

-genomes of thousands of unrelated people with a particular disease are analyzed, usually using microarray analysis -results are compared with people without the disease to identify genetic variations that may increase risk of getting the disease -utilize large scale use of SNP microarrays that probe for 500,000 SNPs to evaluate results from diff people -by looking at SNP changes that occur in people with the disease, scientists can calculate the disease risk associated with each genetic variation using statistics

-Genes are separated by regions of spacer DNA -Genes are on both strands, DNA on both strands can code -Genes on different strands can overlap -If there is the same gene running in the opposite direction on the opposite strand > it codes for a different protein

-human have fewer overlapping genes than simpler eukaryotes

Gene Chips - Microarrays

-oligonucleotides specific for different genes are spotted onto a slide - 64,000 genes on an area the size of a microscope slide coverslip -used for looking at gene expression patterns in genetic diseases -may use probes for only a few specific genes thought to be expressed in different cell types OR may contain probes for each gene in the genome -On this chip, pieces of DNA that will hybridize to specific probes are used to look at different gene expression in different tissues -Take normal cells and tumor cells, isolate mRNA in both, change them into cDNA, and treat them with fluorescent dyes, amount of each cDNA is proportional to amount of mRNA - mix fluorescent cDNAs and wash and let them dry and hybridize and they'll find the oligonucleotide they correspond to - laser and measure the amount of fluorescence in each, which is the output *****the probes are already on the chip/microarray for certain genes you're looking at, so when you add the fluorescent cDNAs, the cDNAs hybridize to the corresponding field gene that encodes their mRNA -yellow means they are expressed, or present in the same probe gene -you have a gene as the probe, whatever hybridizes to it (the color) tells you if the mRNA is expressed in normal or cancer cells -More red > tumor -More green > normal -Yellow has equal amount of mRNA in normal and cancer cells - level of expression of the same gene in both normal and cancer cells -Used to see which genes have been turned on or off in tumor - red is the gene turned on in cancer cells -If an mRNA is present only in normal cells, the probe representing the gene encoding that mRNA will appear as a green dot because only the "green" cDNA have hybridized to it

Some genes in eukaryotes are present in multiple copies - like for rRNA and tRNA -slide set 31 slide 19

-rRNAs 500-5000 copies in tandem arrays -tRNAs 2 to 20 copies in clusters -coding genes themselves are copies -We need multiple gene copies because there is no amplification process for making rRNA/tRNA SO to have enough rRNA for ribosomes we need to have multiple copies of the gene -normal genes can get amplified many times for more multiplication

Gene Families Evolve by Gene Duplication through Unequal Crossing Over Followed by Sequence Drift

-repeated sequences in the genome - transposable elements - increase the frequency of recombination errors like this -Gene families come from unequal crossing over, if chromosomes are misaligned and crossing over occurs, there will be a duplication on one chromosome and deletion on the other -Duplicating a gene doesn't harm anything so you retain the original gene with a second copy that can take on new function - important in evolution of new genes, let evolution shuffle the parts - this is where transposable elements become important because there presence in the genome creates the errors in crossing over leading to new families - when they align w repeat seqeunces it confuses meiosis mechanisms and forms misalignments when crossing over occurs SO VALUABLE FOR EVOLUTION OF THE GENOME

SNPs - single nucleotide polymorphisms (single nucleotide changes)

-resequencing of the human genome in different individuals has identified 10 million SNPs -SNPs can be used to identify gene variants associated with complex disorders (polygenic traits). -A polygenic trait is one whose phenotype is influenced by more than one gene. Traits that display a continuous distribution, such as height or skin color, are polygenic. -Use these as genetic markers and use these in screening too -May be associated or linked to alleles that cause different disorders -Used to find genes that are associated with complex disorders or polygenic traits, each gene having a tiny effect on increasing the disorder

Bacterial genomes

-small -circular -very little repeated DNA -closely packed protein coding genes -lacks introns -polycistronic genes common but do have monocistronic

RFLPs - only used when there is a mutation in the restriction site, this is the only way you'll see a difference if you run 2 different peoples DNA on a gel

-so you have a cloned DNA segment with an RFLP -if there is a mutant allele, in the presence of a restriction enzyme, it will not be able to cut it, so there will be a large band -for a normal allele there will be 2 smaller bands -so if someone is heterozygous, there is 1 large band for the mutant allele and 2 small bands for the normal -someone with sickle cell (that is homo recessive) will have just 1 large band for both mutant alleles -and homozygous dominant will be 2 smaller bands and thats it

Oligonucleotide synthesis

-ssDNA sequences (up to 40 - 50 bases) are easily synthesized. -Synthetic reactions: add one base at a time and you can make things up for a specific sequence. -short, single stranded fragments of DNA that can be used as a DNA probe for alleles that differ by even a single nucleotide, will hybridize only to complementary base sequence, not even to a sequence off by 1 base

Process of PCR

-start with double stranded DNA to be cloned in test tube with primer (present in excess), DNA polymerase, 4 DNA bases (A/G/C/T) and -first you denature the DNA (heat it up 92-95 deg) -Then the primers anneal to the single stranded DNA (when it is cooled down 45-65 deg) -the primers are extended by DNA polymerase (Taq) at the 3' end, the temperature is raised a little 65-75 deg -That is the end of the first cycle - number of DNA molecules is doubled

Northern Blot of Human RNA from Different Organs: Probed with cloned FMR1 gene (mutations in this gene result in mental retardation)

-take RNA from different cells, run it out on agarose gel, and transfer to a membrane paper, and run it on single stranded DNA probe derived from a cloned copy of a gene -In this case, we are probing for the fmr1 gene (fragile x) RNA from different tissues here and this northern blot tells us that mRNA that corresponds to fmr1 is 4.4 bases long, and that there is more in the brain, placentena, etc and very little in the liver -In the heart the message is small > this may be due to alternate splicing of the mRNA *****Bigger fatter band > more rna that corresponds

Screening a gene library by DNA hybridization using a cloned gene as the "probe"

-the probe is a DNA or RNA sequence that is complementary to the target gene/sequence to be identified in a library 1. colonies of the library are overlaid with a DNA binding membrane such as nylon > this transfers the pattern of bacterial colonies from the plate to the membrane 2. Colonies are transferred to membrane, then lysed and denatured with NaOH 3. Membrane is placed in a heat sealed bag with a solution containing the labeled probe; the probe hybridizes with complementary DNA from colonies 4. Membrane is removed from bag and rinsed to remove excess probe then dried; Xray film is placed over the membrane > hybridization of a clone to colony shows up by a spot on Xray film 5. Colonies containing the inserted probe are identified from the orientation of the spots on the original plate, and removed 6. cells that hybridized with the probe are transferred to a medium for growth and further analysis

Gene Families - many protein coding genes in eukaryotes are present in multiple copies - similar but not identical (as with alpha and beta)

-we also have multiple copies of protein coding genes, which tells us how genome functions -A gene family is a group of genes that code for similar but not identical proteins EX hemoglobin -We have alpha and beta gene families for hemoglobin, on different chromosomes, there are 5 copies of alpha that are similar but not identical -Chromosome 16 - alpha gene family -Chromosome 11 - beta gene family -On chromosome 11 (beta) - there are 6 copies of that gene all within 50,000 bp, the basic structure of all the genes are very similar, and the length for all beta chains are the same but have amino acid differences between them -Alpha and beta are so incredibly similar -look for surrounding genes to see if it is part of a gene family, lots of pseudo genes ***pseudogenes -- nonfunctional copies of coding genes - found in both alpha and beta -the number of copies includes the pseudo genes too

Steps in Cloning a Gene

1. Isolated DNA from the organism 2. Cut DNA with Restriction Enzymes 3. Insert DNA fragments into a "Vector" (plasmid) 4. Introduce recombinant DNA (in the form of the plasmid) into host cell E. coli 5. Grow the host and reisolate recombinant DNA clones **to distinguish host cells that have taken up vectors, the vector should carry a slectable marker gene - usually an antiobiotic gene or an enzyme absent from host cell, because then will be obvious when plated on this antiobotic and show they are there

Some Strategies for Screening Libraries

1. Probing using an already cloned gene 2. Isolate the protein, sequence it and make a synthetic oligonucleotide 3. Expression Libraries - antibody detection used

"Shotgun Cloning" approach used by Celera

1. genome cut into small pieces with Restriction Enzymes and cloned 2. clones are sequenced and contigs identified by overlapping end sequences 3. chromosome sequence assembled -Use multiple restriction enzymes, make multiple libraries, and sequence the ends of the pieces, and using this to identify which overlap each other and identify contigs this way instead of physically mapping it, "if 2 pieces have the same end they probably came from same chromosome" -Identified contigs by sequencing the ends then they sequenced the whole pieces and used the computer to put together final chromosome assembly **Computers assemble the sequence of individual contigs into one continuous sequence based on where they overlap/ align (meaning they have the same dna sequence there)

If you have a genetic test to see if you are a carrier of cystic fibrosis and the test comes back negative does this mean your children cannot get the disease?

ANSWER: maybe, maybe not -Difficulty in genetic screening can only detect the known alleles, and we don't know all the alleles that cause the disorder with cystic fibrosis - we know one allele and that accounts for 80% of the people who have it, the other 20% of people who get it don't know where they got it from -Getting back a negative test doesn't mean you're not gonna get it, but a lesser chance, but that u may have a rare allele that can cause it that they didn't test for -If it comes back positive - then you will get it, you have the common allele that causes it

DNA Markers for Genetic Screening -- RFLPs Restriction Fragment Length Polymorphisms

Base changes or small insertions and deletions that vary between individuals can create or eliminate restriction enzyme sites. (as seen in sickle cell anemia testing)

In Most Eukaryotes much of the Genome is repeated DNA sequences

Humans: 3 billion base pairs of DNA -50% repeated noncoding sequences: *most are transposable elements -45% single copy, noncoding sequences -5% codes for genes - this is the total of introns and exons (1.5% exons) ***L1 and Alu are the major transposable elements in the human genome

Genome sequencing based on mapped "contigs" (overlapping genomic DNA clones)

Map-based Cloning: 1. large DNA clones are mapped 2. individual contigs are restriction mapped subcloned and subclones are sequenced (subclones derived from one clones, and then that subclones can be cut up by restriction enzymes and mapped to determine its sequence) -first an entire chromosome is cut into short over lapping sequences most likely by restriction enzymes, -overlapping clones are mapped with in situ hybridization -subclones derived from one of many cloned fragments -each subclone can be characterized by restriction mapping, and this way each subclone will be cut at restriction sites, fragments cloned, and nucleotide sequence determined

Vectors

Plasmids -origin of DNA replication -antibiotic resistance gene -unique restriction sites *Size of the recombinant fragment is limited to ~10 kb Vectors are bacterial plasmids- they're modified for cloning. The basic features of all of them is that they can replicate (origin of replication), antibiotic resistant genes (so you can introduce this without DNA cells recognizing it) and it must have places around the plasmid where restriction enzymes only cut once, and only once. You want it to be open up so it can accept an insert, and the plasmids have been modified where the sites only cut once. -the plasmid vector is removed from host cell and cut by a restriction enzyme, DNA to be cloned is cut with the same restriction enzyme, and then these 2 pieces are ligated, and introduced into bacterial host cells by transformation

White colonies carry a recombinant DNA plasmid Blue ones do not

The cells making beta G turn blue, and if they don't, they're white. The white colonies contain a plasmid with an insertion ability. The blue ones just have the plasmids by themselves. -Original plasmid has lac-z gene to make x-gal and form blue colonies > multiple cloning site is cut > DNA cut with the same restriction enzyme is added and ligased to plasmid > DNA frag inserted anywhere in multiple cloning site, the lac Z gene is disrupted and cannot make beta G > recombinant plasmid cannot metabolize x-gal and will form white colonies

Output of a DNA sequencing reaction using fluorescently labeled bases

The fluorescent tags on the detector and signal system: as you get to longer pieces the peaks go down and the widths become wider and then you can't distinguish the peaks anymore ***ALL OF THIS TECHNOLOGY CAME OUT OF HUMAN GENOME PROJECT

Whats interesting? Why do bacteria have them all?

These are sequences that cut DNA so youd expect it to cut its own DNA which would be lethal, but it makes restriction enzymes and a corresponding methlyase at the same time, which methylates the other strand which blocks cutting. This doesn't happen in humans, so these enzymes are for the bacteria's immune system. Foreign DNA is moved around and if the cell has the restiction enzyme it can cut the foreign DNA up and protect itself from being genetically modified. In the labs- we get rid of this function so DNA can be cloned

Gene Families Evolve by Gene Duplication followed by Sequence Drift

a-globin versus b-globin 50% sequence similarity d-globin versus b-globin 93% same (d is on beta chain) -The globin genes (myoglobin + hemoglobin (a/b)) - the relationships between them and the points at which the gene duplication occurs - based on sequence similarities -You get the timeline by figuring out when the gene gets an amino acid substitution (rate of mutation assuming its constant) and another way is to use the fossil record, knowing where different groups diverged from each other to see what alpha genes they do and don't have -We think these dates are accurate, divergences in gene families don't occur often but over time

Restriction maps

establishes the number of, order of, and distances between restriction sites along a cloned segment of DNA > thus providing information about the length of the cloned gene and the location of restriction sites within the clone.

Recombinant DNA

joining of DNA from different organisms in the test tube •isolation of individual genes (Gene Cloning) •study of gene function and regulation of expression •cloned genes can be sequenced and their base sequences can be modified •transgenic organisms - insertion of cloned genes into animals, plants or bacteria "GENETIC ENGINEERING"

Multiple cloning site

plasmid vectors have this which allows them to have many restriction sites for many restriction enzymes- allows scientist to clone a range of different fragments generated from many common restriction enzymes

How to make cDNA? Use reverse transcriptase- turns RNA to DNA, and the source of this is retroviruses. The retroviruses are RNA viruses that replicate by turning their genome into DNA and inserting it into genomes, (HIV) To clone a mRNA for eurkaryote—isolate total RNA from the cell, add to it a short piece 20 bases long of just T's (oligo dt primer) and they will base pair with the poly A tail. That serves as a primer site for reverse transcriptase site, and it will start the replication . RNA and DNA together now, and RNA H degrades RNA and it only degrates RNA that is attached to DNA. You add this in to take away the RNA but not all, then add DNA poly 1 and it will fill in the spaces and will use the primer to chug along and cut out RNA and replace it with DNA. Add DNA ligase & seal, now you have a DNA piece that coresponds with a messanger. To get into a plasmid, you must manipulate more steps (don't need to know) Just know reverse transcriptase & enzyme Rnase H For HIV- it has RNAse into it (read)

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