Lab Quiz 3 Flashcards
What are VNTRs?
Variable number tandem repeats: repeats of a short DNA sequence that are clustered together and orientated in the same direction
What is DNA fingerprinting
Technique used to identify individuals by examining their DNA. Most fingerprinting methods used today rely on analysis of VNTRs
What is the Tm equation?
Temperature at which 50% of the primer will anneal to the template DNA
Tm (C) = 4(C+G) + 2(A+T)
What does one band in the gel lane tell you?
- You are homozygous
- You are heterozygous, but the bands did not resolve
- You are heterozygous for a deletion of this region (you only have one allele)
What are the PCR components?
- DNA template
- Two single stranded DNA primers
- DNA polymerase
- dNTPs
- Magnesium ions and other salts
What are the steps in PCR?
- Denaturation: reaction is heated to 94 C to separate the two strands of the DNA template
- Primer annealing: reaction is cooled to allow the primers to anneal to their complementary sequences on the template.
- Primer extension: reaction is heated to 72 C and DNA polymerase catalyzes of new DNA strands.
How do you maximize reaction efficiency and specificity for PCR?
- sequences lack inverted repeats, which minimize secondary structure
- Primers are not complementary to one another
- Primers have similar melting temperatures
What are the 3 phases of amplification?
- Exponential
- reagents plentiful
- target doubles each cycle
- Linear
- reagents become limiting
- target does not double each cycle
- when transition from exponential to linear occurs can vary between different reactions
- Plateau
- reagents depleted
- target levels change little with additional cycles
What is end point PCR?
- Is useful for qualitative analyses, such as determining presence/adsence of raget DNA in a sample
What is qPCR
Assesses PCR products during the amplification process
qPCR utilizes a reporter molecule whose level of fluorescence is directly proportional to the amount of amplified DNA in a sample
What are the two type of reporter systems for qPCR?
- Probe-based reporter system: reporter fluorescence when released from quencher by 5→3 exonuclease activity of polymerase
- Dye-based reporter system: fluorescence when bound to double stranded DNA.
What is the threshold cycle (Ct or Cq)
Cycle at which fluorescence for a given sample reaches the threshold and occurs within the exponential phase of amplification.
The higher the starting number of copies of target DNA in a sample, the fewer cycles it takes to reach the threshold.
What are melting curves good for?
A single peak in the melt curve indicates a single DNA amplicon was amplified
