Lab 4 Flashcards
How identical are human genomes, on average?
99.9%
What are the variable regions in genomes called?
Polymorphic
What are transposable elements?
Short DNA sequences (100-1000bps) that can move to other parts of the genome.
What is the Alu element?
A 300 bp transposable element.
Do Alu insertions usually harm a human genome?
Typically they occur in non-functional areas of DNA, such as introns and thus do not have harmful effects. Can have effects if inserted into important genes.
What characterizes the tPA25 Alu insertion?
Dimorphic, phenotypically neutral as it occurs in an intron.
What are the possible ways an individual can be affected by the tPA25 Alu insertion and their corresponding PCR results?
- Homozygous for insertions, PCR produces 400 bp product
- Homozygous for no insertions, PCR produces 100 bp product
- Heterozygous; PCR produces 400 bp and 100 bp products
How are positive ions removed from the lysate?
To remove these positive ions, cell lysate is mixed with Chelex (BIO-RAD, #1421253), a grainy resin with a negatively charged surface which binds to metal ions under alkaline conditions.
How will we limit DNA degredation in this lab in the short and long term?
To limit these activities, cheek cell lysates should be stored on ice until it is used for PCR. For long term storage, lysates can be frozen at -20°C which will keep DNA intact for weeks.
What is a pellet?
Materials collected at the bottom of tube after centrifugation.
What is a supernatant?
Solution remaining after centrifugation, above the pellet.
What does it mean to resuspend a pellet?
Re-dissolve the pellet in a new solution
Is your DNA in the pellet or supernatant?
Supernatant
What is PCR?
Polymerase Chain Reaction (PCR) is a powerful method used to selectively amplify a specific region of a DNA template.
What are the three steps of PCR?
Denaturation, annealing, elongation
What occurs in denaturation? At what temperature?
Template DNA is heated to 95 °C to separate it into two single strands.
What occurs in annealing? At what temperature?
Lowering the temperature to 50 - 65 °C allows Fp and Rp to anneal to template DNA
What does annealing temperature depend on?
Tm (temp at which 50% of DNA is denatured), dependent upon %GC.
What occurs in elongation? At what temp?
Increasing the temperature to 72 °C allows DNA polymerase to extend from the 3’ ends of Fp and Rp
How many cycles does PCR usually occur for?
30-35
Draw what occurs in the first 2 cycles of PCR
look at lab manual
Why is Taq DNA polymerase used?
So the enzyme only needs to be added once
What is the purpose of the PCR buffers?
Sets the correct conditions for primer annealing and Taq polymerase activity
What are the purposes of MgCl2?
- Essential cofactor for Taq DNA polymerase
- May also affect primer annealing and template DNA denaturation
What are dNTPs?
Reagents for DNA synthesis
What does the forward primer do?
Anneals to template DNA strand and gets extended by Taq DNA polymerase
What does the reverse primer do?
Anneals to template DNA strand and gets extended by Taq DNA polymerase
What does Taq DNA polymerase do?
Synthesizes new DNA on template DNA
What is the no-template negative control? What is its purpose?
a reaction is set up without the template DNA. PCR should not work in this sample, and any observation of amplification is indicative of other reagents being contaminated by DNA.
What is the positive control?
A reaction is set up using template DNA which is already known to be amplifiable. The positive control ensures that the PCR conditions used for the experimental reactions were not compromised
