1B- Use of Recombinant DNA Techniques

Question 1

What special molecules does the Sanger method use?

Answer 1

The Sanger method uses dideoxynucleotides (ddNT) for DNA sequencing. These lack the 3’-OH as well as the normal 2’-OH which is found on all DNA.

Question 2

What happens when the ddNT’s are added into the growing RNA chain in the Sanger method?

Answer 2

When the ddNT is incorporated onto the replicating DNA, the next nucleotide cannot add and polymerization is terminated

Question 3

What is added to each tube in the Sanger method?

Answer 3

only 1 of the 4 ddNT’s (ddATP, ddTTP, ddGTP and ddCTP) are added to a tube containing all 4 normal dNT’s, DNA pol, a primer and a template strand of the DNA that is being sequenced

Question 4

During the Sanger process, in the tube, where do the chains terminate??

Answer 4

The chains will terminate at each of the locations in the template strand that is complementary to the ddNT

Question 5

What does the DNA arresting point tell you in the Sanger analysis?

Answer 5

The DNA products will arrest at certain lengths telling you what nucleic acid is at that point, therefore allowing you to see the sequence of DNA on a gel

Question 6

What are oligonucleotide probes?

Answer 6

Oligonucleotide probes (15-20 nucleotides) can be synthesized that are complementary to a DNA sequence that includes a mutation

Question 7

What is the ASO process?

Answer 7

Region of genome with mutation is amplified by PCR –> samples placed onto nitrocellulose paper –> probe added for either normal or mutated gene –> autoradiography to see if probe bound the DNA

Question 8

What is somatic cell therapy?

Answer 8

the therapeutic genes are transferred into the somatic cells (body) of the patient

Question 9

True or False: Somatic cell therapy cannot be passed onto the patients offspring.

Answer 9

True

Question 10

What type of mutations are gene replacement therapies best for?

Answer 10

best for loss-of-function mutations from a missing gene product

Question 11

What are the role of retroviruses in gene therapy?

Answer 11

insert their DNA into the genome (transduction)

Question 12

What is the main benefits of using retroviruses in gene therapy?

Answer 12

1. very effective. 2. no immune response

Question 13

What are some problems when using a retrovirus in gene therapy?

Answer 13

Problems arise if they integrate the DNA in a proto-oncogene or some other bad place.

Question 14

What are the roles of adenoviruses in gene therapy?

Answer 14

a dsDNA virus that is often used for vaccines. They do not integrate into the host genome, so they don’t have the risk of activating a proto-oncogene or something. They just make the protein product that’s missing in the patient.

Question 15

What are some disadvantages to using adenoviruses in gene therapy?

Answer 15

They can be eventually inactivated and they may elicit an immune response.

Question 16

What are the 3 gene blocking therapies?

Answer 16

Antisense, ribozyme and RNAi therapy

Question 17

What types of mutations are gene blocking therapies good for treating?

Answer 17

gain-of-function or dominant negative mutations

Question 18

What is the role of the antisense oligonucleotide in antisense gene blocking therapy?

Answer 18

antisense oligonucleotide binds the abnormal mRNA, preventing its translation into the harmful protein

Question 19

What are some drawbacks to using antisense oligonucleotides in gene blocking therapies?

Answer 19

antisense oligo’s are often degraded before they can reach their target. The antisense oligo might not be able to bind its sequence due to mRNA shape variation

Question 20

What are ribozymes?

Answer 20

Ribozymes are enzymatic RNA’s that can cleave mRNA

Question 21

How can ribozymes be used as a gene blocking therapy?

Answer 21

If they can be made to stab some bad mRNA, then they can be given to seek and destroy that biatch

Question 22

What is the mechanism of action of RNAi’s in gene blocking therapies?

Answer 22

RNAi works a lot like siRNA’s or miRNA’s. Dicer cleaves the dsRNAi, which can then bind to the mutated mRNA and cleaves it by working with RISC

Question 23

How can non-inherited diseases be treated through gene therapy?

Answer 23

To treat such diseases you need to prevent the growth and spread of the disease by getting rid of the things that the disease uses to grow and function

Question 24

What is germline therapy?

Answer 24

Germ cells (sperm or eggs) are modified by the introduction of function genes, which are integrated into their genomes

Question 25

True or False: germline therapies cannot be passed onto the patients offspring.

Answer 25

False

Question 26

What are some reasons why germline therapies arent used?

Answer 26

injected embryos usually die, others develop gnarly problems, it’s hard to distinguish between genetically normal embryos and heterozygous carriers of diseases, and there are ethical issues when you alter a human’s genetic legacy