Biotech ASE
What is the purpose of the tracking Dye?
it is used to visualize the movement of DNA molecules during electrophoresis.
What happens to the speed of migration of DNA fragments when the %agarose increases?
it slows down
what would happen if c DNA was not used when inserting a gene into a plasmid why
it would be recognized by the bacteria --> c DNA =coding dna aka only exons
Developed a method to sequence DNA.
sanger
reverse transcriptase
An enzyme encoded by some certain viruses (retroviruses) that uses RNA as a template for DNA synthesis.
genetically modified foods
An organism whose genetic material has been altered through some genetic engineering technology or technique.
dna fingerprinting
Analysis of sections of DNA that have little or no known function, but vary widely from one individual to another, in order to identify individuals
Worked with Rosalind Franklin and received the Nobel Prize as well as Watson and Crick.
wilkins
Transformation
(genetics) modification of a cell or bacterium by the uptake and incorporation of exogenous DNA
sample preparation for gel electrophoresis
Mix the samples of DNA with the 6X sample loading buffer (w/ tracking dye). This allows the samples to be seen when loading onto the gel, and increases the density of the samples, as it has glycerol added to increase the density to greater than water. This causes the dye and DNA sample to sink into the gel wells. Remember the wells are like little cups, open at the top and walls on each side.
What is the charge on DNA molecules?
Negative
loading dye
Mixed with restriction fragments before running a gel. intended to weigh down the fragments so they sink in the well and provide visual confirmation of movement.
Figured out that the proportion of A's was equal to the number of T's and the proportion of C's was equal to G's.
chargaff
acts as a vector to carry a new gene into a bacterium or pollen grain
Plasmid
how does PCR work
Thermocycler changes temperature repeatedly to create correct conditions for DNA replication, warm splits, cool primer stick, cooler new DNA
what technique should be used to determine if someone can donate and organ to someone else
blood typing
removes the DNA from the gel and applies it to paper for further analysis
blotting
who do we use hybridization probes
for a known gene, the complementary DNA is created with a molecule being attached that glows or is radioactive. Then the film is used used to detect what fragment has the probe on it
taq polymerase
found in thrum aqueous from hot spring bacteria so it will not denature when doing PCR
Took x-ray pictures of DNA and got dissed when it came to recognition.
franklin
Did the first successful transformation experiment
friffith
combining different pieces of DNA together, typically from different species
recombinant DNA
what technique should be used to create a tomato that is a vaccine for a disease
recombinant DNA
what cell can you not use for dna for electrophoresis
red blood cell
what type of cloning was dolly produced by
reproductive cloning
2 ways cloning can be done
reproductive cloning and therapeutic cloning
The site of translation.
ribosomes
The enzyme that helps DNA unzip and helps complimentary bases match up in transcription.
rna polymerase
Word describing the fact that when DNA replicates, half of each strand is old half is new.
semiconservative
Marker DNA is used to create what type of graph, that is then used to find the sizes of fragments on the same gel?
standard curve on semi-log paper
These are created when a restriction enzyme cuts the DNA and leaves single stranded overhangs.
sticky ends
why are sticky en useful
sticky ends are useful for gene splicing (which is used to create genetically modified organisms)
The thing that tells the ribosome to stop translating and release the polypeptide.
stop codon
Staining the Gel in electrophoresis steps
• Place the gel in the staining tray containing warm diluted stain. • Allow the gel to stain for 25-30 minutes. • To remove excess stain, allow the gel to destain in water. • Replace water several times for efficient destain.
safer alternatives to ethidium bromide
∙ Methylene Blue ∙ BioRAD - Bio-Safe DNA Stain * Ward's - QUIKView DNA Stain * Carolina BLU Stain
atheneum bromide
stains the dna but its a powerful mutagen
what technique should be used to making dna from an eyelash more accessible to dna testing
pcr
an enzyme that cuts DNA at a specific sequence of bases
restriction enzyme
*need to make sure bacteria have recombinant plasmid
* do a test to see bacteria have plasmid for example put gene for lactose so if there is no gene in the plasmid it will stay blue but if there is it will stay white *or if there is gene then they will die (only some) and if there is not they will live *LacZ gene on plasmid produces digestive enzyme : Lactose (X-gal) → blue thus leading to blue colonies *insert foreign DNA into LacZ gene breaks gene : lactose (X-gal) → blue thus leading to white colonies *white bacterial colonies have recombinant plasmid
anode end
* it is the positive en dot which the negative dna will migrate to through h the agarose gel
somatic cell nuclear transfer
*-somatic cell: cell from the body -nuclear: having to do with the cells nucleus -transfer: movement of one thing to a different place *1st isolate the somatic cel, then remove the nucleus and all DNA from an egg cell, then transfer the DNA from the isolated somatic cell into the now DNA free egg cell, now you let the cell become a blastocyst and from here the blastocyst can be used fro therapeutic cloning or for reproductive cloning *this creates a clone and it starts by using a diploid cell... while natural reproduction starts with a haploid cell and it creates genetically unique organisms.
microassay
*A glass slide carrying thousands of different kinds of single-stranded DNA fragments arranged in an array (grid). A DNA ______ is used to detect and measure the expression of thousands of genes at one time. Tiny amounts of a large number of single-stranded DNA fragments representing different genes are fixed to the glass slide. These fragments, ideally representing all the genes of an organism, are tested for hybridization with various samples of cDNA molecules. *a chip is prepared with many different specified DNA segments; mRNA from cells is then placed on the chip and the amount of binding is used to determine which mRNAs are present in the cell; essentially it is a bunch of in situ hybridizations at once
standard curve
*A graph relating a measured quantity to concentration of the substance of interest in "known" samples *can be generated by producing a series of solutions of known concentration, which can be used to determine the concentration of an unknown solution
southern blotting
*A technique that enables specific nucleotide sequences to be detected in a sample of DNA. It involves gel electrophoresis of DNA molecules and their transfer to a membrane (blotting), followed by nucleic acid hybridization with a labeled probe. *Through a series of steps, DNA that has been separated by electrophoresis is applied to a membrane of nylon or nitrocellulose.
Intron problem
*Gene sequence must be made without introns *Take mRNA after introns are cut out and use reverse transcriptase to make copy DNA which is DNA without intron sequences. *Bacteria can't process introns later.
adult stem cells
*Induced pluripotent stem cells aka IPSC -are pluripotent -can be taken from bone marrow -adult cells not undifferentiated -this is the turning back of differentiated cells -created by turning on or by introducing stem cell genes, or by using chemicals *somatic stem cells -multipotent -are in each of us, such as the stem cells in the bone marrow->can turn into blood cells -they are in teeth, heart, gut, bones, blood, blood vessels, and muscles -special considerations: they exist in low abundance in the body
human cellular cloning, therapeutic cloning
*It is created by 1) somatic cell nucleus and an enucleated e.g. are fused 2) a zygote is created 3)the zygote becomes a blastocyst 4)stem cells can be harvested and then differentiated into different types of cells such as pancreatic cells, nerve cells, or muscle cells *can be used to get replace organs/parts/or tissues to treat diseases such as spinal injuries -the replaced organ/part/tissues will not be rejected because it has the same marker proteins *it doesn't allow the cloned zygote to develop into an offspring instead the zygote dies after the stem cells are removed in the blstocyst stage *this can create Embryonic stem cells that are pluripotent and that won't be refused by the body
What does LB stand for
*Lysogeny broth and it is the food for the bacteria *luria broth and it provides nutrients for bacteria to grow
runninng the gel in electrophoresis/applying the current
*Place the cover on the electrophoresis chamber, connecting the electrical leads. Connect the electrical leads to the power supply. Be sure the leads are attached correctly - DNA migrates toward the anode (red). When the power is turned on, bubbles should form on the electrodes in the electrophoresis chamber. *After the current is applied, make sure the Gel is running in the correct direction. Bromophenol blue will run in the same direction as the DNA.
selectable marker
*Plasmid has both "added" gene & antibiotic resistance gene *If bacteria don't pick up plasmid then die on antibiotic plates *If bacteria pick up plasmid then survive on antibiotic plates *selecting for successful transformation
what does RFLP stand for
*RFLP= restriction fragments length polymorphism *restriction fragments: the fragments of DNA that were cut by restriction enzymes *length: the length of the restriction fragments *polymorphism: this refers to the different shapes/lengths of the fragments
melting the agarose
*because agarose is insoluble at room temperature it must be heated until it is clear *gently swirl the solution periodically when heating to allow all the grans of agarose to dissolve *be careful when boiling -- the agarose solution may become superheated and may boil violently if it has been heated too long in a microwave oven
genetic engineering
*The technology entailing all processes of altering the genetic material of a cell to make it capable of performing the desired functions, such as producing novel substances. *Genetic engineering is the deliberate, controlled manipulation of the genes in an organism with the intent of making that organism better in some way. This is usually done independently of the natural reproductive process. The result is a so-called genetically modified organism (GMO). To date, most of the effort in genetic engineering has been focused on agriculture. *if you are going to engineer DNA & genes & organisms, then you need a set of tools to work with --> whats the right tool --> whats the right skill
PCR aka polymerase chain reaction
*To perform this reaction you need a DNA that has bee extracted from cells -this DNA can come from skin cells, saliva, or hair follicles *it uses: buffers->to keep the PH the same, primers->to tell the polymerase were to begin copying (will either attach to the first or the 2nd site), polymerase enzymes, and free nucleotides *PRC works by -denaturation: heat is used to separate two strands of the DNA to be copied (no helicace) -anneling: the primers are short sections of DNA with a specific base sequence complementary to a portion of the DNA to be copied. The primers anneal or bid to these portions, this is called hybridization. This marks the starting point for replication -Extension/Elongation: DNA polymerase replicates the DNA by riding along the unpaired strands using free nucleotides to create the missing strands -repeat many times *a laboratory method of making numerous copies of DNA from a very small amount
vectors (overview)
*a vehicle that can transport the DNA piece into a host cell *there are mechanical vectors and biological vectors *A short piece of DNA capable of replicating on its own when inside a bacterial cell *any tool used to insert foreign DNA into a cell this includes plasmids, viruses, gene guns, micropipettes, etc.
advantages and disadvantages of alternatives to ethic bromide
*advantages: they are inexpensive, less toxic, no UV light required, no hazardous waste disposal *disadvantages: less sensitive, more dna needed on gel, longer staying/destaining time, blue stains smear are much harder to see
how to map a plasmid
*after cutting the plasmid up with several restriction enzymes *use electrophoresis to determine the sizes of the fragments produced *use the fragment sizes to piece together the plasmid map with all restriction sites labeled
competent cells
*after icily have better gene uptake as competent cells aka a dna passes easier thus it is competent *you can make competent cells by using calcium chloride and a heat shock *cells undergoing rapid growth are more competent
agar vs agarose
*agar: from seaweed is food for bacteria to grow upon *agarose: sugar gel for doing electrophoresis
how to make agarose gel
*agarose gel is prepared by combining agarose powder and a buffer solution using a flask for boiling *prepare casting tray *combine the agarose powder and buffer solution. use a flask that i several times larger than the volume of the buffer *melting the agarose *pouring the gel *let it cool *put gel into electrophoresis chamber *add enough electrophoresis buffer to cover the gel to a depth of at least one mm make sure each well misfiled with buffer
how thick should the agarose gel is
*agarose is measured in % which is grams per 100 ml of solution *a 7% gel vs a 3% gel : the thicker is going to be easier to hold without breaking but it will be slower for molecules to go through and hard for big molecules to make it
pouring the agarose gel
*allow the agarose solution to cool slightly to about 60C and then carefully pour the melted agarose solution into the casting tray avoiding air bubbles *each of the gel combs should be submerged in the melted agarose solution *when cooled the agarose polymerizes foreign a flexible el which should appear lighter in color when completely called (30-45 minutes) *carefully remove combs and tape
transgenic organisms
*any organism that has its own DNA plus working genes from another species *the genes must be working/functioning so they can make proteins *transgenic organisms have recombinant DNA *an organism that has been engineered to carry functional genes from another organism
methyl tags and histone coiling and acetyl tags
*are epigenetic tags, the methyl tags turn genes off while histone coiling has to do with coiling up the DNA, and if the DNA is coiled up it is off and it if uncoiled it is on *histone: proteins that hep organize DNA , in Stem Cells DNA is loosely wrapped around histones ready to spring into action and in differentiated cells DNA is tightly wrapped around histones *methyl tags are used to science genes by blocking their transcription *acetyl tags: they loosen interaction between DNA and histones
Induced pluripotent stem cells aka IPSC
*are pluripotent *can be taken from bone marrow *adult cells not undifferentiated *this is the turning back of differentiated cells *created by turning on or by introducing stem cell genes, or by using chemicals *they have potential as therapy since they can become any cell, and it is less expensive than Embryonic Stem cells *results of reprogramming IP stem cells aren't perfect yet this is because there are distinctive methyl tags on IP stem cells that are not on other stem cells and as a result IP stem cells are not totally the same as stem cells
the results of making transgenic bacteria
*as the cell reproduces all of its offspring will have a clone or identical copy of the recombinant DNA *as a result you only have to make a few transgenic bacteria and reproduction will create thousands/millions for you *ALL THE BACTERIA WILL TRANSLATE THE DNA INTO THE DESIRED PROTEINS... the transgenic bacteria will make the protein
where do restriction enzymes cut
*at the recognition sequence/restriction site which is usually 4-8 nucleotides long. this sequence is also palindromic and so it cuts DNA ever time at that sequence so one ddba strand would create many fragments *used to cut up dna for DNA fingerprinting via electrophoresis
Bacterial examples of GMO / Genetic Engineering
*bacteria: -scientist have genetically engineered bacteria to enhance the ability to metabolize oil -this could be used to clean up oil spills
reverse transcriptase use
*because bacteria don't recognize introns son one must make exons only, make RNA, make transcription RNA -->to make RNA and make reverse transcription of RNA using reverse transcriptase
why do bacteria make good transgenic organisms
*because they grow quickly meaning they'll make a lot of protein, and they reproduce quickly meaning that soon you'll have lots of them making the desired protein
plasmid mapping
*coming up with the locations of the cleavages on the plasmids
What is cDNA?
*complementary DNA Single-stranded DNA synthesized in the laboratory using messenger RNA as a template and the enzyme reverse transcriptase. *copy of mRNA containing protein coding sequence of a gene without the introns.
recombinant DNA
*connected pieces of DNA from different sources *transgenic organisms have recombinant DNA *combining different pieces of DNA together, typically from different species
blastocyst
*created 5 days after fertilization * a blastocyst is special because it is when an early embryo is formed and as a result the todipotent stem cells can either be harvested or the blastocyst could be implanted into a mom in order to create a clone.
action of restriction enzymes
*cut DNA at specific sequences restriction site which is a symmetrical "palindrome" and the cutting produces protruding ends sticky ends
epigenome
*deals with the different ways that our genes are regulated *An Epigenome consists of a record of the chemical changes to the DNA and histone proteins of an organism; these changes can be passed down to an organism's offspring. *The epigenome comprises all of the chemical compounds that have been added to the entirety of one's DNA (genome) as a way to regulate the activity (expression) of all the genes within the genome. The chemical compounds of the epigenome are not part of the DNA sequence, but are on or attached to DNA ("epi-" means above in Greek). Epigenomic modifications remain as cells divide and in some cases can be inherited through the generations. Environmental influences, such as a person's diet and exposure to pollutants, can also impact the epigenome. *It shapes the physical structures of our genome
differentiation
*differentiated cells:cells that are fully developed, meaning that they are already developed into the type of cel that it wants/has to be *without differentiation we'd be a blob of zygotes but due to differentiation were are able to develop different functioning body parts, making us complex. *the epigenome is important in the process of celular differentiation during embryological development because all the cells in an organism have the exact same DNA but different genes turn on and off in different cells to create the variety of cell types that exist in a multicellular organism.
types of stem cells
*embryonic stem cells *somatic stem cells *induced pluripotent stem cells aka IPS cells
biological vectors
*examples include: -using viruses for bacteria, animals, plants -using plasmids for bacteria
southern blotting/probes
*find
making a transgenic organism all steps
*first isolate the desired DNA gene by using restriction enzymes that cleave the DNA at the desired places *then put the DNA piece into a vector which will transport the desired DNA piece into the host cell
making a transgenic organism step one
*first you must isolate the desired DNA/the desired gene that is in the chromosomes in the nucleus *you can isolate using restriction enzymes
embryonic stem cells
*from a blastocyst/embryo created thru therapeutic cloning -pluripotent -cells are identical to the cloned person *they can be donated to science, kept for ever, or killed *from a blastocyst/embryo -identical to no one -fully adult cell is differentiated -pluripotent *A zygote is totipotent until after a few divisions, then it becomes pluripotent
what are the main issues with gene therapy?
*gene delivery is the hardest thing in gene therapy because -the vector must AVOID getting attacked by the immune system -the vector must be able to TARGET the right cells -the vector must be able to INTEGRATE its "message" into the host cells genome -the vector must ACTIVATE the genes it codes for *also not all diseases can be treated with gene therapy, they must be single gene diseases, the genes must be known, we must understand the disease, and adding a correct copy of the missing gene must be able to cure the disease
other examples of GMO/ Genetic Engineering
*goats: -scientist have genetically engineered goats with a gene from spider to produce spider silk in goats milk -this silk can be used for artificial tenants and ligaments and this could also be used to create bullet proof vests *pigs: -scientist have engineered pigs with genes that will allow them to extract more phosphorous from their food -this would reduce the amount of phosphorous in animal poop-> and this would reduce water pollution
agricultural examples of GMO/Genetic Engineering
*golden rice: -scientist genetically engineered rice planes with a gene to produce beta-carotene and vitamin A -It would help people in developing countries get vitamin A-> and as a result the people could avoid going blind *salmon: -scientist genetically engineered genes from to fish into salmon that increase growth rate x3 -It would help the salmon get to the market faster -> this would make more money *soy beans: -scientist genetically engineered soy beans with a protein that makes them resistant to herbicides -as a result farmers can kill weeds with herbicides with out damaging the soybeans *plants: -scientist genetically engineered plant genes in order to control sodium ions -This would allow scientist to be planted and harvested in salty areas
bacteria transformation
*host takes foreign DNA and expresses foreign gene; gene in lab is for resistance to ampicillin
heat shock
*increase in temperature in order to denature things or lead to plasmid up take or denaturing of DNA
RILP analysis of DNA fingerprinting
*individuals DNA is different
applications of gel electrophoresis
*it can be used to pinpointing genes responsible for hereditary diseases, confirming family relationships and individual identities, solving crime scenes, and styling the diversity of genes within population
Epigenetics
*it deals with the different ways our genes are regulated *the epigenome consist of molecular systems that can turn on/off and regulated gene expression -the signals that can turn the genes on or off can be instigated through direct contact, hormonal signals, or by signals nearby *all cells have the same DNA as a result of mitosis, but different genes turn on/off in different cells creating different cell types that is why this epigenetics important in cell differentiation in embryological development -epigenetic marks are used to separate different types of stem cells from each other by turning genes on/off *epigenes can also change during ones lifetime due to exposure to different environments *epigenetic's gene switches may be inheritable *it is not just the DNA that is changed but it is also the things surrounding the DNA -controls the expression of genes
artificial embryo twining
*it is a low tech way to make clones that mimics the process to make identical twins *in nature: the embryo splits into two-> sort of like binary fission *artificial embryo twining does the same but in a patri dish instead of incise a mom creating a two cell embryo
gel electrophoresis
*it is a way to sort and measure the DNA strands. -this technique is also useful for separating other types of molecules such as proteins *Electrophoresis is how we push DNA strands through the gel filter by adding an electrical current, with a negative charge at the holes and a positive charge at the other end *to do gel electrophoresis you must -1st place the DNA at the holes at the end of the gel -then you must turn on the electricity to watch the strands move -afterwards the result must be stained so that the groups of DNA can be visible to the naked eye *shorter fragments move faster than longer fragments and as a result fragments of the same size will clump up together *a standard technique that separates molecules such as DNA on the basis of size by using electricity to separate pieces within a gel matrix known as agarose gel. Since each individual has unique DNA this technology allows you to visualize differences based on how DNA separates within the gel.
e coli in transformation
*it is grown in a lure broth in order to ge million cells aka a colony *it is common in gut and widely studied because they reproduce fast *they do not have a nuclear envelope surrounding dna --> no nucleus: all goes in 1 chromosome *hav eplamsids
agar/agarose
*it is like making j-ello *agarose is seewed sugar --> is is a linear polymer extracted from seaweed *sweetened agarose gels have been eaten in the far east since the 17th century *agarose was first used in biology when robert koch used it as a culture medium for tuberculosis bacteria in 1882
biotechnology
*it is the manipulation of living organisms and their life processes to manufacture products that improve the quality of life. This is a very broad term and it includes everything from using yeast to make bread to using DNA to solve a crime. *developing better procedures *pieces that we use and how we use them
dna ladder
*lane marker *Used as a control to tell you how big the DNA is.
problems with bacteria not knowing introns
*make exons only, make RNA, make transcription RNA *to make RNA and make reverse transcription of RNA using reverse transcriptase
hybridization radioactive probe
*makes something complementary, going finding it and lighting it up
lambda dna
*marker DNA, famous virus and helps to know the size while doing DNA fingerprinting *DNA already cut with two restriction enzymes
retrovirus
*once a retrovirus infects a cell its RNA travels to the cells nucleus-> as a result the viruses RNA must be converted to DNA before the genes are activated: the virus contains enzymes to do this job *the Human Immunodefency virus which causes aids is a retrovirus *it is a viral vector *retroviruses only infect dividing cells so they cannot target cells tat have stopped dividing. It is possible to target specific cell types by engineering proteins not he virus surface *THe DNA made from the virus' RNA integrates the host cell's genome in random locations -> this lead to a chance that the retro virus' RNA integrates into a place were it disrupts another gnu resulting in possible side affects such as tumors
ligase
*paste or put together *enzyme "seals" strands --> bonds sugar-phosphate bonds --> covalent bond of DNA backbone
plasmid
*plasmids can be used as vectors-but they are considered non viral vectors *in nature bacteria use plasmids to transfer and share genes with one another *they can carry larger ends than viruses and most don't trigger an immune response but they are less efficient in getting the genes into the cells *an extra piece of circular DNA found in bacteria separate from the main circular chromosome *they are bad at reproducing within cells, they can be rejected by the cell membrane , and they are not as stable with effective gene expression *they copy and are in bacteria and serve for transformation *they also serve as a vector
electrophoresis equipment
*power supply *gel tank *cover *electrical leads the red is + and black is - *casting tray *gel combs --> used for making wells
uses for PCR
*producing copies for DNA finger printing *studying ancient DNA samples *identifying diseases and mutations *Detecting bacteria and viruses *finding out who the daddy is *solving a crime *to detect mutations *to recombine
advantages and drawbacks of viral vectors
*pros -they are good at targeting and entering cells -some target specific cell types -they can be modified so that they can't replicate and destroy cells *cons -they can carry a limited amount of genetic material, so some genes are to big to fit into some viruses -they can cause immune responses in patients (patients could get sick, or the immune system could block the virus from delivering the gene/it may kill the cells once the gene has been delivered) -can lead to inflammation
Pros and cons of GMOS
*pros: -some scientist say its safe -lower price for food -increased farmer safety due to the reduction of pesticides, herbicides, insecticides and other harmful chemicals -it could lead to end world hunger -more food -it could be engineered to help deliver scarce but necessary vitamins such as the golden rice does -it is faster to produce crops with desired traits than by doing selective breeding (and non gmo selectively bread crops can end up tasting bad or looking weird when cooked) *Cons: -some scents don't rust it because it involves combining genome -could accidentally create toxic plants -it is not trusted -it can and has been leading to pest resistance -creating GMO's is a chemical baked approach rather than a knowledge based approach (selective breeding) -it is looked down upon because it tryes to better God's creation -it involvers the ethical issue that people want to try to act like God and this is wrong .
restriction enzymes
*proteins that can cleave/cut DNA at certain places *they were originally found in bacteria and their original use was to kill viruses within the bacteria or at least preventing the bacterial virus from reproducing within the bacteria *an enzyme that cuts DNA at a specific sequence of bases *discovered in 1960 *they evolved in bacteria to cot up foreign DNA and they served for protection against viruses and other bacterial DNA trying to enter --> bacteria protected their own DNA by methylation and by not using the base sequences recognized by the enzymes in their own DNA * they are proteins that cut the phosphodiester back bone of DNA which can be remade by ligase and when they cut they either create sticky ends or blunt ends
making a transgenic organism step two
*put the DNA/ the desired gene piece into a vector
RFLP analysis
*restriction fragment length polymorphism; the method for preparing a DNA fingerprint *restriction fragment length polymorphism analysis; differences in DNA fragment sizes cut by specific restriction enzymes, which are used as markers on genetic maps, to help identify the source of DNA and to determine the presence of specific alleles or genes *RFLP is restriction fragment length polymorphism. This is the idea that each person's DNA would cut up into different sized fragments. Thus the pattern produced by electrophoresis of those patterns would be unique to the person.
preparing the casting tray when making agarose gel
*seal the edges of the casting tray and put in the combs. place the casting tray on a level surface. none of the gel combs should be touching the surface of the casting try
plasmids
*small, self-replicating prokaryotic circular DNA molecules *insert DNA sequence into plasmid *vector = "vehicle" into organism *Original plasmid is call a cloning vector *transformation *insert recombinant plasmid into bacteria --> bacteria make lots of copies of plasmid *grow recombinant bacteria on agar plate --> clone of cells = lots of bacteria *prokaryotic circular DNA that is 1,000 to 1,000,000 nucleotides *it is extrachromosomal so its outside the main chromosome *it is used by bacteria to exchange with other bacteria via conjugation in order to confer antibiotic resistance for bacterium *can be part of gene therapy where plasmid attempts to carry Dna into adult specimen * circular dna outside the main chromosome used as a vehicle to introduce genes into bacteria
human being cloning, reproductive cloning
*steps 1) somatic cell nucleus and an enucleated e.g. are fused 2) a zygote is created 3)the zygote becomes a blastocyst 4)the blastocyst is implanted into the uterus 5)pregnancy leads to the clone baby *this clones the whole organism * SCNT and artificial embryo twinning are types of human being cloning
how fast will dna migrate in gel electrophoresis
*strength of the electrical field, buffer, density of the gel and the size of the dna will play a part in hotfoots the dna moves *small dna moves faster than larger DNA thus allowing gel electrophoresis to separate by size *within an agarose gel linear DNA will migrate inversely proportional to the log10 of their molecular weight
potency of stem cells
*the ability of stem cells to become something -totipotent= can become all type of cells -pluripotent=can turn into many types of cells -multipotent=it can turn into a lot of cells but not into all cells
what are transposons
*they are unstable DA aka moving DNA that jumps from one chromosome location to another chromosome location *they are jumping elements in the genome which have the abnormality of reproducing weird activities in the genes near their new position to which they move -for example the jumping of these gene elements that have integrated in a gene involved in corn pigmentation results in a mosaic pattern phenotype on corn *the transposons have potential as vectors because they are effective in achieving stable gene expression of ends introduced into animal cells, and by using enzymes we can eliminate tho problem of the jumping of the transposons *transposons can move through -replicative transposition: a new copy of transposons appears at a new site and the original element stays at the old location -conservative transposition: it does not have a copy, therefore it is the original one that moves around
Junk DNA
*this is the non coding DNA sections of DNA between genes that are about on.1% is unique for every individual- that is about 3 million letters
gene therapy
*this technique is used to cure disorders such as hemophilia, CF, blindness, deadly bubble boy disease, and HIV by changing the genome -must be single gene diseases -must have knowledge on disorder -will adding a normal copy of the gene fix the issues? -can the gene be delivered to the affected tissue (example: for CF gene can be delivered with an inhaler) *gene delivery must be able to function properly in order to do gene therapy, lethal un predicted outcomes may occurs (such as jesse's inflammation), when using viruses as vectors we must be carful with immune responses. *Not all diseases can be treated by gene therapy, and for a gene therapy to be successful vectors must target, integrate, activate,and avoid *a method of treating genetic disorders by inserting a functional copy of the missing or non functional gene into target cells
mechanical vectors
*tools such as: -a gene gun used for plants -micropipettes used for animals
stem cells
*undefined cells with the ability to develop into a number of different types of cells *stem cells play an important role from embryo to adult hood: -development begins with a zygote which is the only totipotent stem cells , and the first few divisions after that are still totipotent stem cells until some genes begin differentiation... this differentiation begins after then pluripotent blastocyst *stem cells are very powerful because they can lead to regeneration of body parts such as in salamanders and in starfish, as a result stem cells have a potency in cloning
mapping a plasmid
*unlike mapping a chromosome where we map the gens position relative to each other using crossing over data, mapping a plasmid is mapping the relative locations of restriction sites on a plasmid for use during genetic engineering
transforming procedure
*use plasmids with amp to transform e lacking that gene *have a control group to make sure E won't grow in agar and ampicillin if they are -amp^R *amp sensitive e cells in log cells are transferred to cold call solution *amp r plasmid is added to the experimental cells only *cells are heat shocked and some competent cells will take up plasmid *the treated cells are spread on agar plate with ampicillin *the cells are incubated *only colonies of icily with resistance that have been transformed will grow
probe
*used to "light up/identify" specific genes *Often probe is radioactive or fluorescent so when exposed to film it can be detected.
ethidium bromide
*used to stain the gel *Ethidium bromide binds to DNA and fluoresces under UV light, allowing the visualization of DNA on a Gel. • Ethidium bromide can be added to the gel and/or running buffer before the gel is run or the gel can be stained after it has run. *Ethidium bromide is a powerful mutagen and is moderately toxic. Gloves should be worn at all times. *ethiudium bromide requires an ultraviolet light source to visualize *it is a carcinogen and it is hard to get
name uses for transgenic organisms
*we could make better plants, more resistant to pesticides therefore reducing insecticides; this could lead to more crops/food and eventually to the end of world hunger *putting breast cancer genes into mouse zygotes in order to test drugs on mouse and hopefully find the cure to cancer *growing bacteria with human insulin genes in order to get insulin for diabetics
Hot to create a GMO tomato
1) cleave the desired gene 2) place the desired gene inside a vector ... in the tomatoes case it was a Bt gene obtained from bacteria *the first two steps are the steps to make a transonic organism 3)add the vector back to a bacteria 4) grow the bacteria 5)put cells from the desired plant with the growing bacteria so that the bacteria can insert their modified genes into the plant 6)kill the losers
steps to clone dolly
1) donated egg cell from a different type of sheep than the nucleus donor 2) the donated egg cell is enucleated aka the nucleus ifs removed 1) donor skin cell taken from a the mammary gland of the nucleus donor... a different type of sheep that the egg cell donor 2) the donor cells are starved: the scientist simulated the cells death without killing the cells in order to try to reset the genes into making them believe they they are a zygote 3) the enucleated egg cell and the donor cell (arrested growth cycle) are fused together... this is the Somatic Cell Nuclear Transfer step .... aka this is the fusion of the adult cell DNA and the empty egg 4) electrical pulses 5) cell division 6) the embryo is then implanted into the surrogate mother which will give birth to Dolly *THIS WAS SCNT *DOlly was the wrist successfully cloned mammal from an adults sheep cell
DNA finger printing
1) extract DNA from crime scene evidence which could be hair, blood cells etc. And also extract DNA from suspects 2)amplify your sample of the crime scene evidence by using polymer ace chain reaction aka PCR which is a process that makes millions of copies of your DNA samples 3) cut the DNA into pieces with restriction Enzymes 4) then separate the pieces by size by pulling them through a gel by the process of gel electrophoresis in which short pieces travel faster than longer fragments 5) create a picture where the DNA fragments stopped in the gels and compare: the matching DNA fingerprints indicate DNA from the same person. *DNA finger prints can also be used to determine hereditary DNA things *a method of identifying an individual based on their DNA sequence using electrophoresis and proves; most commonly used to present crime scene evidence, determine paternity, and help diagnose genetic disorders
Genetic engineering steps for making a transgenic Bacteria
1) isolating/ removing the desired gene aka piece of DNA by using restriction enzymes 2) removing the plasmid from the bacterial cell 3) cleaving the plasmid and the gene for HGH 4) splicing the human gene into plasmid 5) form a recombinant plasmid 6) put the recombinant plasmid back into the bacteria 7) the bacteria will produce the desired protein (HGH)
Transcription starts at this end of the template DNA molecule.
3'
The end of the mRNA that first attaches to the ribosome.
5'
how many loci do they analyze
6-12 aka they don't analyze all dna because thats hard
tracking dye
A material used to visualize the movement of DNA molecules during electrophoresis.
primer
An already existing RNA chain bound to template DNA to which DNA nucleotides are added during DNA synthesis.
dilution formula
C1V1=C2V2 where C is concentration and V is volume
loading the gel in electrophoresis
Carefully place the pipette tip over a well and gently expel the sample. The sample should sink into the well. Be careful not to puncture the gel with the pipette tip.
pluripotent
Cells that are capable of developing into most, but not all, of the body's cell types
cDNA
Complementary DNA. DNA produced synthetically by reverse trascribing mRNA. Because of eukaryotic mRNA splicing, cDNA contains no inrons.
allows the gene to be permanently sealed into the plasmid
DNA ligase
dna sequencing
Determining the exact order of the base pairs in a segment of DNA
serial dilution
Dilution of a substance several times by the same amount each time
colonies
Distinct populations of bacteria
The molecule that is put on the 5' end of the mRNA.
GTP cap
dna with another indicator molecule that attaches to a fragment or other piece of DNA to indicate where that specific DNA code is
Hybridization probe
In the lac operon, what does the repressor bind to?
In the absence of lactose, the Lac repressor binds to the operator and keeps RNA polymerase from transcribing the lac genes.
Thermocycler
Instrument that cycles through several temperature cycles over a period of time, DNA & DNA Polyermase placed in thermocycler will produce copies of DNA
screening agent
Make sure inserted plasmid is recombinant plasmid aka that it takes up recombinant gene.
What cuts phosphodiester bonds at the recongition sequence
Restriction Endonucleases
what technique should be used to solve a crime when the only evidence is a flake of skin
PCR and DNA fingerprint
blunt ends
Restriction fragments with no overlapping ends and that never combine with another type of DNA
selecting agent
The condition or conditions in an ecosystem that determine which individuals (ones best suited to take advantage of the conditions) are more likely to survive.
allows the scientist to see the DNA sample being loaded into the well and determine when to turn off the electricity during electrophoresis
Tracking dye
antibiotic resistance gene
a gene usually inserted in a plasmid that provides special resistance to a bacteria against an antibiotic --> these are the winners that will survive unlike the losers that will die
well
a hole in gel where we place a sample
dna fragments
a piece of dna
Vector
a piece of dna or something used to transfer genes
any tool used to insert foreign DNA into a cell this includes plasmids, viruses, gene guns, micropipettes, etc.
a vector
The molecules that bind to the enhancer DNA region
activator
What is the typical gel substance used to do electrophoresis?
agarose gel
gel of this provides pores or DNA to move through at different speeds based on size
agrose
allows the scientists to determine if a gene has been added to a plasmid or whether a plasmid has entered the bacterium
antibiotic resistance gene
Performed experiments with bread mold to support the one gene-one protein hypothesis.
beadle and tatum
why is important to have a symmetrical palindrome for a recognition sequence
because so a cleave to sticky ends
buffer
buffer is going to keep the pH constant during gel electrophoresis because if the ph changed the h+ charges would be pulled to the negative cathode and would set up a ph gradient in the box
The molecule created when mRNA is copied using reverse transcriptase
cDNA or copy DNA
growing cells in vitro aka in the laboratory in a culture medium such as a petri dish or liquid culture. Cell culturing allows scientist to test how different chemicals/drugs/medicines etc. affect human cells without needing human test subjects.
cell culture
what technique should be used to growing cells in a petri dish to see drug could affect human show a
cell culture with stem cells
The series of 3 bases on the mRNA that codes for a certain amino acid.
codon
What are the plastic instruments that are used to make the wells?
combs
hybridization
complementary dna binds to a gene and holds another molecule to "light up/identify" a specific sequence of DNA
how do we map plasmids
cut plasmid in several different ways. each digest should have one enzyme or multiple enzymes that produce fragments that can be pieced together to make a plasmid map.
what technique should be used to determine paternity
dna fingerprint
The enzyme that helps complimentary bases match up during replication.
dna polymerase
what technique should be used to determine evolutionary relationships of species
dna sequencing
separation of DNA fragments by size
electrophoresis
what technique should be used to method that separates pieces by size or charge
electrophoresis
applies the energy to separate the DNA fragments based on size
gel box
how do we do blotting
gel is placed on paper and then large weights are placed on top to have it bond to paper
a method of treating genetic disorders by inserting a functional copy of the missing or non functional gene into target cells
gene therapy
what technique should be used to treat cystic fibrosis by inserting genes that create proteins to break down mucus
gene therapy
what technique should be used to make a crop of corn that is resistant to drought
genetic engineering
what technique should be used to use a plasmid gene gun or virus as a vector
genetic engineering
what technique should be used to use dna ligase to seal sticky ends
genetic engineering
what technique should be used to determine whether someone is predisposed to rbreast cancer
genetic testing
encourages bacterium to take up DNA and be transformed
heat shock
Figured out that DNA was the genetic material by coloring N and P in viruses.
hershey and chase
The type of chromatin that is not available for transcription
heterochromatin
The proteins that DNA wraps around as it coils up.
histones
a worldwide effort by scientist in the 1990's to identify the 20,000 to 25,000 human genes and to sequence of all of the DNA found along our 23 chromosomes. The project was completed in 2003. This knowledge can help in the understanding of the genetic causation of disease and to develop new technologies for diagnosing and treating disease.
human genome project
DNA ladder standard
it is like a control group and the Inclusion of a DNA ladder (DNAs of know sizes) on the gel makes it easy to determine the sizes of unknown DNAs.
types of bonds that hold together nitrogenous bases
hydrogen bonds
glycerol in gel electrophoresis
it is used to sink it to the bottom
tracking die in electrophoresis
is used to estimate smaller fragments
amp ^r
it is having the gene for ampicillin resistance unlike -amp^R which does not have resistance gone but it can be transformed by adding the gene
what technique should be used to determine theater a person has klienfelters
karyotype
what technique should be used to determine theater a person has turner syndrome
karyotype
what technique should be used to determine whether nondisjunction has occurred
karyotype
The enzyme that joins together DNA fragments such as those connected in gene splicing.
ligase
The type of RNA that carries the code for synthesis of a protein.
mRNA
allos for us to determine the size of fragments on electrophoresis by showing known sized pieces and their movement
marker dna/ dna ladder
What is added to chromatin to make it heterochromatin and also protects bacterial DNA from its own restriction endonucleases?
methyl group
what technique should be used to transfer very small amounts of liquid
micropipetting
The charge on the DNA molecule
negative
which type of restriction enzyme would work best for gene splicing one that makes a blunt cut or one that makes a jagged cut
one that makes a jagged cut because the base paring would hold the sticky ends together ... that is if the same enzyme is used. And because trying to joint two blunt cuts is hard since there is no base paring involved to make them stick to each other
what technique should be used to determine the genotype of parents for hemophilia
pedigree analysis with punnet square
The type of bond that forms between amino acids.
peptide
Small circular pieces of DNA found in bacteria.
plasmids
loci
point on the chromosome where the gene is found
two ways DNA can be "labeled" to know how far it has gone
probes, or ethium bromide
What are two characteristics of all recognition sequences
reciprocal palindrome 4-8 nucleotide bases
The pallandrome that a specific restriction endonuclease would cut.
recognition sequence
what technique should be used to use of these creates sticky ends
restriction enzyme digest
creates sticky ends for engineering or fringiest for electrophoresis analysis
restriction enzymes
cuts DNA at specific sequences
restriction enzymes
how does electrophoresis work
restriction enzymes cut DNA at different recognition sites on different people. Electrophoresis separates the segments by pulling them through an agarose gel where negative DNA moves to positive side of box
Viruses are sometimes used to get plasmid DNA into a bacterium. What kind of virus would be used?
retroviruses
knockout gene
specific gene is targeted and disrupted and the animals are studied for any behavioral changes that might occur as a result
how do we clone mammals
take the nucleus out of an egg cell and replace it with full set of diploid dna from the parent. the clone will have identical DNA to the parent but the telomeres are worn down.
The four nucleotide region within the promoter that the transcription factors bind to
tata box
What is the part of the DNA that the RNA polymerase binds to for transcription?
tata box
lina hesse
technician and illustrator for a colleague of koch was the first to suggest agar for use in culturing bacteria
In the Sanger method, what stops the replication of sequences?
the dideoxy-nucleotide analogs have no 3'-OH group to continue DNA synthesis.
once a human gene has been cleaved and then added to a plasmid, which enzyme would be added to complete the splicing process
the enzyme DNA ligase
how do we use restriction enzymes
the enzyme when placed with DNA sample cuts it every time it recognizes the same palindromic sequence.
Wells
the little wholes in the gel in electrophoresis
What is the goal of the Sanger method?
to determine the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA.
What is the purpose of antibiotic resistance in genetic engineering?
to kill the losers and keep the ones with the plasmid
wha tis the process where the plasmid changes a bacterium called
transformation
an organism that has been engineered to carry functional genes from another organism
transgenic organism
Two men most famous for discovery of the double helix.
watson and crick
allows the DNA to enter the gel from the side at the right depth
wells
when do we do gel electrophoresis
when doing DNA fingerprinting, analysis of plasmids, determining sizes of fragments, mapping plasmids
