L3- DNA Techniques Flashcards
What is the function of restriction endonucleases?
They are bacterial enzymes that recognize and destroy foreign DNA.
How do bacterial enzymes differentiate endogenous DNA from foreign DNA?
Bacterial DNA is methylated by endogenous enzymes, whereas foreign DNA is not. That way, bacterial enzymes can discriminate between endogenous and invading DNA.
Restriction endonucleases generate DNA fragments that can be fractioned by gel electrophoresis. What is the main determinant of mobility of these fragments?
Size. Smaller fragments move through the gel faster than larger fragments.
Which technique is most appropriate for differentiating very large DNA fragments (100-1000kb)?
Pulsified gel electrophoresis.
What do Southern blots separate?
DNA fragments.
Once they are run on a gel, how are DNA sequences of interest isolated?
DNA fragments of interest are hybridized with a radioactive 32-Phosphorous-labeled probe that is complimentary to the fragment.
A scientist inserted the CFTR gene into the genome of a mouse. To detect if the gene was integrated, s/he decided to run a Southern blot. List the steps of Southern blotting in order.
- Cleave genomic DNA with restriction enzymes 2. Perform gel electrophoresis 3. Transfer fragmented DNA from gel to nitrocellulose membrane 4. Hybridize DNA of interest (in this case, CFTR gene) with labeled probe 5. Expose membrane to film and develop.
A physician wants to test a patient for hemoglobin S (sick cell hemoglobin). The only test available in the lab is a Southern blot. Is it possible for the physician to diagnose the patient?
Yes. Point mutations whose location in the DNA sequence are known can be detected using a Southern blot if the base substitution or deletion is in a sequence recognized by a restriction endonuclease (this would lead to a loss of a restriction site).
A physician wants to test a patient for a newly discovered genetic disease. The nature and location of the mutation causing the disease is not yet known. The only test available in the lab is a Southern blot. Is it possible for the physician to diagnose the patient? If yes, under what condition?
Yes, but only if the mutation causing the disease is closely associated with a novel sized restriction fragment.
What is the location and nature of the mutation that causes sick cell hemoglobin?
The mutation involves a change in codon #6 of the beta-globin gene from GAG (glutamate) to GTC (valine).
The mutation causing alpha1-antitrypsin deficiency does not create or destroy a restriction enzyme site. Which laboratory technique is most appropriate for detecting this disorder?
Allele-specific oligonucleotide probe. Note that these probes can hybridize to DNA only when the nucleotide sequence of the normal and mutant alleles are known.
True or False. When DNA is cleaved into restriction fragments, the pattern of fragments on a blot varies from one individual to another according to where and how many repeat units are located within the genome.
True. This is due to the polymorphic variability of the human genome.
Which technique is most appropriate to detect the size and amount of specific mRNA molecules?
Northern blots.
Which technique is most appropriate to detect the size and amount of a specific protein?
Western blots.
What is the function SDS-polyacrymalide gel with regards to Western blotting?
Different proteins carry different charges. SDS is an anionic detergent that denatures proteins and essentially gives them all the same charge. This allows for the separation of peptides according to size instead of charge.
