Cycle 10 Flashcards
Q: What is the purpose of PCR?
A: To amplify a specific region of DNA, creating many copies for further analysis.
Q: What are the main reagents required for PCR?
A: DNA template, primers (forward & reverse), Taq polymerase, dNTPs, MgCl₂, and buffer.
Q: Why is Taq polymerase used in PCR?
A: It is derived from thermophilic bacteria, allowing it to withstand high temperatures without denaturing.
Q: What are the three main steps of PCR, and their temperatures?
A:
1) Denaturation (95°C) – DNA strands separate.
2) Annealing (55°C, varies) – Primers bind to target DNA.
3) Extension (72°C) – Taq polymerase synthesizes new DNA strands.
Q: What is RT-PCR, and what is its main purpose?
A: Reverse transcription PCR (RT-PCR) is used to measure mRNA levels (gene expression/transcript levels) by converting mRNA into cDNA, which is then amplified.
Q: What enzyme is required for reverse transcription?
A: Reverse transcriptase, an enzyme derived from retroviruses.
Q: How is mRNA selected for reverse transcription?
A: Using an oligo(dT) primer, which binds to the poly-A tail of mRNA.
Q: What are the two key functions of reverse transcriptase?
A:
1) DNA polymerase activity – Synthesizes DNA from an RNA template.
2) RNase activity – Degrades RNA in RNA-DNA hybrids to create double-stranded cDNA
Q: Why do we use cDNA instead of mRNA for experiments?
A: mRNA is unstable and cannot be amplified by PCR, whereas cDNA (made from mRNA) can be used for PCR-based studies.
Q: How is PCR used for DNA profiling?
A: PCR amplifies short tandem repeats (STRs), which are unique to individuals and can be compared in forensic and paternity testing.
Q: What are STRs (Short Tandem Repeats)?
A: Repeated DNA sequences (4–6 base pairs long) found in non-coding regions of DNA, used for identification.
Q: How do STRs vary between individuals?
A: The number of repeats differs, making STRs highly polymorphic and useful for distinguishing DNA samples.
Q: How many STR loci does the FBI use for DNA profiling?
A: 13 core STR loci, analyzed through CODIS (Combined DNA Index System).
Q: How can PCR determine biological sex?
A: By amplifying the AMEL gene:
1) XX individuals show one band (shorter AMEL gene on X chromosome).
2) XY individuals show two bands (longer AMEL gene on Y chromosome).
Q: What is an electropherogram?
A: A graphical representation of STR fragment sizes after gel electrophoresis or capillary electrophoresis.
Q: How do you interpret an electropherogram?
A: Each peak represents an STR allele. One peak means homozygous, two peaks mean heterozygous for that locus.
Q: What is the role of a control in DNA profiling?
A: A known DNA sample is run alongside the experimental samples to ensure accurate results.
Q: How is it possible to express a human gene in bacterial cells?
A: The cDNA (not genomic DNA) of the human gene is inserted into a bacterial plasmid, allowing expression of the gene without introns.
Q: Why must cDNA, not genomic DNA, be used for bacterial gene expression?
A: Bacteria lack the machinery to process introns, so cDNA (which lacks introns) is necessary.
Q: How is human insulin produced using bacteria?
A:
1) mRNA for human insulin is extracted.
2) Reverse transcription creates cDNA.
3) cDNA is cloned into a bacterial plasmid.
4) Bacteria express the human insulin protein.
5) Insulin is purified and used for treatment.
Q: What does CRISPR stand for?
A: Clustered Regularly Interspaced Short Palindromic Repeats.
Q: Where does CRISPR originate from?
A: CRISPR originates from the adaptive immune system of bacteria.
Q: What is the role of the Cas9 protein in CRISPR?
A: Cas9 is an enzyme that cuts DNA at a specific location to enable gene editing.
Q: What is the function of Guide RNA (gRNA) in CRISPR?
A: gRNA guides the Cas9 protein to the correct DNA sequence for editing.
