pcr

Question 1

What does PCR allow you to do?

Answer 1

allows you to amplify a speicfic DNA sequence out of pool of DNA containing millions of bases

This works by the new DNA being created serving as the template in the next round of replication.

Question 2

What is taq polymerase?

Answer 2

A DNA polyerase that comes from a species of bacteria resistant to high temperatures

Question 3

What is required for pcr along with the polymerase?

Answer 3

1. 2 primers bind to the sequence of interest on opposite sides
2. dNTPS ( A,C,G,T)
3. buffer with MgCl2 ( Mg++ is required for Taq polymerase to function)
4. template DNA ( in our experiment that is genomic DNA isolated from bacterial colonies)

Question 4

What are the steps of PCR?

Answer 4

1. denaturing at 95 degrees
2. annealing at 50 -60 degrees ( this is optimized based on the primers you are using)
3. extension at 72 degrees without destroying the enzymatic activity of the polymerase.

Question 5

How should primers be designed

Answer 5

with 40-60 percent G/C content, start and end with 1-2 G/C pairs, melting temp of 50-60 degrees, placed at the ends of the sequence to be amplified, and must be oriented to “face “each other

Question 6

Describe the types of mutations

Answer 6

Base substitution Silent – mutation does not result in a change to the amino acid sequence
Missense – mutation results in an amino acid change Nonsense – mutation results in a premature stop codon Frameshifts – insertions or deletions, alters the reading frame resulting in an altered amino acid sequence downstream of the mutation – and possibly a premature stop.