Flashcards in L3- DNA Techniques Deck (33):
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?
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.
1. 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?
Which technique is most appropriate to detect the size and amount of a specific protein?
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.
What kind of molecules are used as probes to detect proteins of interest during Western blotting?
The probes are antibodies specific for target proteins (whereas for Southern and Northern blots, the probes are nucleic acids that are complimentary to a nucleic acid sequence of interest).
What is the first step of the polymerase chain reaction (PCR)?
Heat denaturation of the double-stranded DNA fragment that is to be analyzed (90 degrees Celsius).
A scientist wishes to amplify a fragment of DNA using PCR. However, the scientist does not know the DNA sequence of the regions that flank the fragment of interest. Is it still possible for the scientist to amplify the DNA fragment of interest?
No. The sequence of the flanking regions must be known so that appropriate primers may be selected. Without probes that are complimentary to the flanking regions, the DNA of interest will not be amplified.
What is the special feature of the DNA polymerase used in polymerase chain reactions (PCR)?
It is heat stabilized.
Which technique is most appropriate to amplify a sequence of RNA and how does it work?
RT-PCR. A reverse transcriptase converts the DNA into a single-stranded cDNA molecule, which can then be amplified using standard PCR techniques.
A scientist decides to use the single-strand conformation polymorphism (SSCP) technique to determine if his DNA sample contains mutations. What is the main determinant of gel migration using this technique?
Conformation of the DNA single-stands. If a mutation is present, the conformation of the single-strand will be altered, thereby causing the band pattern to differ from that of the normal single-strands.
The Sanger method is used for DNA sequencing and involves dideoxy-NTPs (dd-NTPs). What is the structure and function of dd-NTPs?
dd-NTP's lack both the 2' and 3' hydroxyl groups on the ribose. Since the 3'-OH group on the ribose is required for chain elongation, incorporation of dd-NTP's will block chain elongation.
Which technique is most appropriate for determining the base sequence of a DNA fragment?
The Sanger method (see syllabus for detailed explanation).
Which technique is most appropriate for determining if a protein interacts with a particular DNA fragment?
DNA band shift assay.
Which technique is most appropriate to determine the boundaries of protein-DNA interactions?
DNase I footprinting assay.
What is the function of the enzyme DNase I in DNase I footprinting assays?
DNase I digests DNA. Regions of DNA bound to protein will be protected from digestion. As a result, their corresponding bands will be absent when the DNA is run on a gel.
What are vectors?
Vectors are DNA molecules that replicate autonomously in a host cell. They are designed to accommodate the insertion of foreign gene sequences.
After a vector is incorporated into a cell, how can recipient cells be sorted from non-recipient cells?
Vectors usually contain genes that confer resistance to particular drugs. Therefore, only recipient cells will be able to survive once the drug is added to the growth medium.
What is the difference between genomic libraries and cDNA libraries?
Genomic libraries are constructed from genomic DNA (introns and exons) whereas cDNA libraries are constructed from mRNA (exons only).
What is a plasmid?
A plasmid is a circular bacterial chromosome that replicates independently of the host chromosome.
Which of the following technique provides a more precise determination of DNA-protein interactions: DNA band shift assays or DNase I footprinting assays?
DNase I footprinting assays.
A scientist wants to determine if a certain transcription factor is present in brain tissue. Which DNA technique is most appropriate to accomplish this task?
DNA band shift assay.