Bio: Chp. 9, Power notes and Other work

PCR steps

1. Denaturation- Heat to 90 degrees Celsius the container to separate DNA 2. Annealing(binding)- cool the container to 55 degrees Celsius and bind primers to opposite ends of the DNA segments 3. Extension- heat the container to 75 degrees Celsius. Polymerase bind until strand is completely copied, and the cycle continues

Describe two ways in which DNA fingerprinting is used.

1- to prove someone's innocence 2- to prove kinship for immigration test

clone

An organism that is genetically identical to the organism from which it was produced

Who developed knock-out mice?

Mario R. Capecchi

What type of vector is most commonly used?

The most common type of vector that they use is a virus

genomics

study of genomes

Describe the general process used to make bacteria that have recombinant DNA.

(1) They cut both the DNA and plasmid with the same restriction enzyme so they will have complimentary sticky ends. (2) The plasmid opens and adds the new DNA to itself. The complimentary sticky ends bind in a process called ligation.

Gene splicing steps

1- remove plasmid and cut using restriction enzyme; remove DNA from human cell and cut using same enzyme to create complimentary sticky ends. 2- Put together and plasmid will take in human gene 3- Place plasmid back into bacterial cell 4- Allow bacterial cell to grow to larger amounts; extract proteins

human genome project goals

1- to map and sequence all of the DNA base pairs of the human chromosomes 2- to identify all the genes within the sequence

What does bioinformatics do?

-compares genomes of different lengths -stores, shares, and finds data -predicts the function of proteins -finds the basis of molecular disease

Cons of genetically modified foods

-could cause allergies -invasive species -lose biodiversity -effect on humans longer term is unknown

Uses of DNA fingerprinting

-evidence in criminal cases -paternity tests -immigration requests -studying biodiversity -tracking genetically modified crops

What does proteomics do?

-it studies the interaction between proteins -studies evolution by studying the similar proteins in species -studies what proteins cause human diseases

Pros of genetically modified foods

-more food -healthier food

There are 4 major questions/factors that must be considered before using gene therapy.

1- Could the condition be corrected by adding one or a few functional genes? 2- Do you know which genes are involved? 3- Do you understand the biology of the disorder? 4-Can you deliver the gene to cells of the affected tissue?

What is gene sequencing used for?

1- comparing diseases and affects of medications 2- study when spec

Experiments for best CF vector

1993- Vector Used: Adenovirus Successful?: no Conclusion: The appropriate dose of adenovirus causes an immune response. Find a better way for the virus to enter the cell. 1995- Vector Used: Liposome Successful?: no Conclusion: Effects don't last long. Needs to improve on delivery and its permanence. 1998- Vector Used: Adeno-Associated Virus Successful?: no Conclusion: This vector is more promising. They need to learn more about how the virus infects cells.

Who is "Dolly"? What made this clone special compared to the previous two experiments?

A cloned sheep. She brought cloning into the limelight and started the conversation on cloning and they used an adult cell. Normally they use embryonic cells.

gene splicing

A type of gene recombination in which the DNA is intentionally broken and recombined using laboratory techniques.

What is a viral vector?

A virus, like the flu

homeotic genes

Any of the genes that control the overall body plan of animals by controlling the developmental fate of groups of cells.

How might cloning of an animal be similar to and different from the cloning that happens in nature?

Artificial clones of animals are used for research, but natural clones happen by chance. In both instances the clones could be clones or the parent; asexual reproduction and/or artificial twinning.

Explain the use of bone marrow in gene therapy and why it is valuable?

Bone marrow contains many stem cells. These stem cells can be used to treat disorders, especially blood disorders.

Which disorders are best for gene therapy and which are poor candidates?

Candidates with one gene that is dysfunctional are the best and the worst are candidates with multiply dysfunctional genes and environmental factors.

What is a virus?

Chunks of DNA wrapped around a protein and sometimes covered in a membrane

Describe three reasons for cloning and give an example from each.

Cloning animal models of diseases- reduces the time for making transgenic animal models for research- Ex: cloning mice for genetic mutation research. Cloning to make stem cells- to help repair organs and tissues of individuals. If not from oneself their body may reject it.- Ex. Oregon health and science university for to make cloned embryonic stem cells. Reviving endangered or extinct animals- to bring back extinct animals or to help endangered populations grow- Ex. scientist in 2009 cloning endangered goats.

Exons

Coding segments of DNA.

recombinant DNA

DNA that contains genes from more than one organism

what are the materials needed for PCR? Describe each and the function of each.

DNA- to be copied DNA polymerase- to build new strands Large amount of free nucleotides- to create the new strand 2 primers- to show were the copying process starts thermal cycler- temperature gage test tube- even heat distribution

Sanger sequencing

Dideoxynucleotides halt DNA polymerization at each base, generating sequences of various lengths that encompass the entire original sequence. Terminated fragments are electrophoresed and the original sequence can be deduced.

How is gene therapy different from drug-based approaches?

Drug-based approaches only treat the symptoms and not the source.

How is it possible that 2 people with the same genome could be different?

Environmental factors play a role into what a person is like and looks like. For example, one twin may have gotten more nurturance in the womb.

restriction enzymes

Enzyme that cuts DNA at a specific sequence of nucleotides

Why might noncoding regions of DNA outside of genes be more variable than coding regions of DNA?

Everyone has the same or almost the same coding DNA. The major differences lie in the noncoding DNA.

What are the 4 main challenges of gene therapy?

Gene delivery and activation- Gene NEEDS to go to the proper cell because it could cause major health problems in the patient. Immune Response- could cause the immune system to reject the vector causing major illness or even death. To prevent this doctors; introduce the vector outside the body into the cells, give the patient drugs to suppress their immune system for a little bit, or lower the dosage. Disrupting important genes in targeted cells- could stitch itself into the wrong place, for example, it could stitch itself into the gene that regulates cell growth and cause cancer. Commercial Viability- very expensive and the success rate is low

How might genomics and proteomics help researchers predict how a gene a medial treatment might affect cells in different tissues?

Genomics is the study of DNA sequences. You could study the DNA of a person not affected by a medicine and one that was; then study the differences in their DNA sequences. Proteomics is the study f proteins that resulted from the genome. Studying the proteins allows us to know what proteins cause what, and different proteins will react to medical treatments differently.

How does genomics rely on DNA sequencing?

Genomics is the study of a genome. In order to study a genome you need to know the order the genes are in. DNA sequencing is the finding of the order of the genes.

What are advantages to viral vectors?

Good at targeting and entering the DNA, can target specific types of cells, and they can be modified so they can replicate and destroy cells.

Who was the scientist that made the first clone?

Hans Adolf Eduard Driesch

What process was used to create the first clone?

He separated the one cell into two by shaking it and they developed separately.

How did Mullis solve the problem of high heat destroying the polymerase?

He used a different polymerase, from bacteria, that could withstand the high heats.

What information must you know before starting gene therapy?

How many genes are mutated, what tissue is being affected by the dysfunctional proteins, and what role do those proteins play?

What leads to viruses that may be able to 'jump' from one species to another?

If a cat, for example, contracts two diseases and one of the diseases can infect humans as well. Some of the genes for the disease that does infect humans cross with the one that doesn't, and now you have two diseases that infect both cats and humans.

How is genetic technology used to track the spread of disease?

If two people have the virus with the same genetic sequence then scientists can tell they got it from the same place. Doing this allows scientists to determine who else was infected and so they can be treated.

How do different restriction enzymes produce different DNA fragments from the same DNA molecule?

It depends on how many restriction sites a restriction enzyme finds. One enzyme may find 3 restriction sites and another may find 6 on the same DNA fragment.

Why might so many different methods be needed to study DNA and genes?

It depends on what you are studying. For example, you could be studying gene expression or studying changes in DNA sequences.

What important fact did Hans Adolf Eduard Driesch experiment show?

It showed that each embryonic cell has there own DNA and can grow into individuals separate from each other.

knock-out mice

Mice genetically engineered to disable a particular gene, and used to create strains of mice with specific traits

Is cloning an organism the same as cloning a gene? Explain.

No, because cloning an organism requires all the genes to be copied exactly. If there is one mess up the whole thing fails. Copying genes there is less room for error.

Introns

Noncoding segments of nucleic acid that lie between coding sequences.

How are pedigrees and DNA testing used to together in genetic screening?

Pedigrees test the chances of you or other people in your family having a disorder by seeing who had a disorder and who didn't. After that they do a DNA test to see if you have proteins that are linked to having a disorder. Genetic screening is used to find if you have a disorder and/or if you could pass it on to your offspring.

What are plasmids?

Plasmids are small loops of extra DNA that aren't part of the chromosome. Plasmids contain genes for things like drug resistance and can be passed between bacteria.

Artificial Embryo Twinning

Purposely dividing growing ball of cells. When this occurs naturally it forms twins.

List the 6 different types of vectors.

Retrovirus, Adenovirus, Adeno-Associated virus, Herpes simplex virus, liposome, and naked DNA

Explain the 4 qualities that vectors must have?

Target the correct cell because it could be the gene for hair growth and it goes to your heart cells. It needs to be able to integrate into the cells so it doesn't because disposed of. It needs to activate in the gene, which means it needs to be able to produce its proteins. It also needs to be nonharmful to the subject.

gene therapy

The insertion of working copies of a gene into the cells of a person with a genetic disorder in an attempt to correct the disorder

What does male mating do? (How/why)

The male's offspring will either have the gene or not. The reason why is because not all of his reproductive cells have the active gene, may have the inactive one.

DNA fingerprinting and probability

The more DNA fragments being tested the less of a probability that the fingerprint will match someone.

Why is the offspring of asexual reproduction a clone?

The offspring of asexual reproduction is an exact copy of the parent.

Why is this 'jumping' a problem?

The other species will not have the ability to fight off the disease because they have never encountered it before.

How does gel electrophoresis separate DNA fragments?

There are two electrodes on either side of the gel. One is negatively charged and the other is positively charged. DNA is negatively charged so, when turned on, it moves to the positive side. There are little pores in the gel. The smaller fragments can easily pass through the pores so they will be reach the positive side more quickly. Since the larger strands have a harder time passing through the gel they stay near the negative for longer.

Suppose you cut DNA. You know that you should find four DNA fragments on a gel, but only three appear, and one fragment is very large. Explain what happened.

There was a mutation in the DNA leading to it not being cut.

How are DNA fingerprints and restriction maps similar? Different? Explain.

They are both the same process with the gel and restriction enzymes. Fingerprint mapping is used to decode regions of DNA not on gene sequences; restriction maps are used to figure out the length of DNA fragments.

How are the genetic engineering processes of making transgenic organisms similar to and different from crossbreeding?

They are similar in the way that it is to organisms creating a new organism. They are different because transgenic genes are specifically chosen and added into the genome. Crossbreeding is more up to chance if the offspring will have the desired trait.

Why are the advantages of non-viral vectors?

They can carry large genes and they don't trigger the immune system.

What are disadvantages of viral vectors?

They can only carry a set amount of genetic material. They can cause problems like; the patient becoming sick or the immune system may block the virus from reaching the cell

How does gene therapy work?

They inject the virus into the person so the virus can deliver the gene to the proper cells. Once in the cell, the cell's RNA makes the new proteins and those proteins carry out their jobs.

What key question did Mario R. Capecchi's work raise?

This brought up the question, if we can replace a working gene with a mutated gene can we do the reverse?

What is the goal of gene therapy?

To help diseases tissue and organs work properly.

Would a mutation in a gene always be detectable by using restriction maps? Why or why not?

Yes, if you compare it to a normal restriction map. You can compare the differences.

PCR requires DNA polymerase from bacteria that live in hot springs. Why can't DNA polymerase from organisms that live in cooler temperatures be used in PCR?

You have to heat the DNA and some DNA polymerases will be destroyed if they are out in too high of a heat.

genetic engineering

a cloned gene from one organism put into another organism

Pharming

a combination of the words farming and pharmaceuticals

gene editing

a highly precise type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of an organism using engineered nucleases

What was the first-ever clone?

a sea urchin

DNA microarrays

a tool that allows scientists to study many genes, and their expression at once

Where do restriction enzymes come from?

bacteria

list the four ways in which scientists can manipulate DNA

chemicals, computers, bacteria, and gel electrophoresis

How is the cloning of genes different from cloning cloning of mammals?

cloning mammals is much more difficult because you are coping the whole organism. Copying a gene only requires copying a segment of DNA. There is also less room for error.

genetically modified organisms

crops that carry new traits that have been inserted through advanced genetic engineering methods

blunt ends

cuts are straight through both DNA strands at the line of symmetry

gene sequencing

determining the order of DNA nucleotide in genes or in genomes

restriction map

diagram that shows the lengths of fragments between restriction sites in the strand of DNA

transgenic plants

genetically modified food or to make vaccine/medicines

Natural Clone

occur without man's help, such as identical twins, asexual reproduction

artificial clone

occurs when man helps an organism reproduce

Summarize each of the success stories of gene therapy thus far

immune deficiencies- they take blood stem cells from the patient. Add retrovirus to them to deliver the proper copy of the gene. Doesn't cause the immune system to flare because it was treated outside of the body and that means it also only target desired cells. First trials of SCID cause leukemia but now the chances are slim for getting leukemia after treating SCID. During ADA treatment no one developed leukemia and after a while, they didn't need more shots. Plus this was treated outside the body. Hereditary blindness- has the potential to slow or reverse vision degeneration from old age. The retina is easy to access. It won't affect the immune system, and it won't spread throughout the body. Hemophilia- this disease prevents the blood from clotting. After some patients received Factor IX to their liver their bodies had produced some Factor IX and had fewer bleeding incidents. Blood Disease- some trials to treat beta-Thalassemia took stem cells from the bone marrow, treat the stem cells with retrovirus to give it the beta-hemoglobin gene, and turned it back to the cell. This lead to a rise of red blood cells in the patient. Fat metabolism disorder- first approved gene-therapy treatment in Europe. They used an adeno-associated virus to deliver a protein that breaks down fat in the blood. Cancer- Used herpes simplex 1 virus to treat melanoma. Treatment T-VEC has been modified to (1) not create cold sores; (2) kill cancerous cells; (3) teach the patient's immune system to kill cancerous cells. It is injected straight into the tumor. They then burst in the cell. Parkinson's disease- gave patients retroviral virus in small areas of the brain and the patients gained better motor control afterward.

Describe the differences between in vivo and ex vivo.

in vivo- to inject the patient with the vector, aiming at the target cell ex vivo- put the cells in a culture, injecting the culture with the vector, then putting the cells back into the patient.

RNA interference

introduction of double-stranded RNA into a cell to inhibit gene expression

how does DNA microarrays work?

it is chips with many dots in a grid formation and the complimentary DNA would be dyed and put back into the microarray

Somatic Cell Nuclear Transfer (SCNT)

method of reproductive cloning in which genetic material is transferred from an adult somatic cell into an unfertilized, enucleated egg

immune cells

modify immune cells to target specific cells and destroy the antigens.

transgenic animals

more resistant to modifications; genetic modification is passed down to offspring; test disorders, knock-out mice

homologous recombination

process that results in genetic exchange between homologous DNA from two different sources

Where do restriction enzymes cut?

restriction sites

Vector

something that delivers DNA

Triple-helix-forming oligonucleotide gene therapy

target DNA sequence to prevent its transcription. Sends single-strand DNA piece to bind with a gene's two DNA strands; this makes a triple-helix structure that can not be transcribed by mRNA.

Ribozyme Gene Therapy

target mRNA transcripts. Ribozyme act like scissors. They separate and destroy mRNA encoded for mutant protein.

Spliceosome-mediated RNA trans-splicing

targets and repairs mRNA transcribed from the mutated gene causing a disorder; doesn't replace dysfunctional gene

polymerase chain reaction (PCR)

technique that allows molecular biologists to make many copies of a particular gene

enucleation

the removal of the nucleus from an egg cell

proteomics

the study and comparison of all the proteins that result from an organisms genome

sticky ends

the uneven ends of a double-stranded DNA molecule that has been cut with a restriction enzyme

bioinformatics

the use of computer database to organize and analyze biological data

transgenic bacteria

used to create proteins for other organisms, like insulin.

What are non-viral vectors?

vectors that are not viruses, like plasmids.

How is the disease diagnosed?

watch for symptoms and do a genetic test

gene silencing

whole regions of chromosomes are shut off while the same regions in other cells remain active