Flashcards in 1B- Use of Recombinant DNA Techniques Deck (26)
What special molecules does the Sanger method use?
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.
What happens when the ddNT's are added into the growing RNA chain in the Sanger method?
When the ddNT is incorporated onto the replicating DNA, the next nucleotide cannot add and polymerization is terminated
What is added to each tube in the Sanger method?
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
During the Sanger process, in the tube, where do the chains terminate??
The chains will terminate at each of the locations in the template strand that is complementary to the ddNT
What does the DNA arresting point tell you in the Sanger analysis?
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
What are oligonucleotide probes?
Oligonucleotide probes (15-20 nucleotides) can be synthesized that are complementary to a DNA sequence that includes a mutation
What is the ASO process?
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
What is somatic cell therapy?
the therapeutic genes are transferred into the somatic cells (body) of the patient
True or False: Somatic cell therapy cannot be passed onto the patients offspring.
What type of mutations are gene replacement therapies best for?
best for loss-of-function mutations from a missing gene product
What are the role of retroviruses in gene therapy?
insert their DNA into the genome (transduction)
What is the main benefits of using retroviruses in gene therapy?
1. very effective. 2. no immune response
What are some problems when using a retrovirus in gene therapy?
Problems arise if they integrate the DNA in a proto-oncogene or some other bad place.
What are the roles of adenoviruses in gene therapy?
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.
What are some disadvantages to using adenoviruses in gene therapy?
They can be eventually inactivated and they may elicit an immune response.
What are the 3 gene blocking therapies?
Antisense, ribozyme and RNAi therapy
What types of mutations are gene blocking therapies good for treating?
gain-of-function or dominant negative mutations
What is the role of the antisense oligonucleotide in antisense gene blocking therapy?
antisense oligonucleotide binds the abnormal mRNA, preventing its translation into the harmful protein
What are some drawbacks to using antisense oligonucleotides in gene blocking therapies?
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
What are ribozymes?
Ribozymes are enzymatic RNA’s that can cleave mRNA
How can ribozymes be used as a gene blocking therapy?
If they can be made to stab some bad mRNA, then they can be given to seek and destroy that biatch
What is the mechanism of action of RNAi's in gene blocking therapies?
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
How can non-inherited diseases be treated through gene therapy?
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
What is germline therapy?
Germ cells (sperm or eggs) are modified by the introduction of function genes, which are integrated into their genomes
True or False: germline therapies cannot be passed onto the patients offspring.