In vitro methods (PCR)

Question 1

PCR

Answer 1

amplification of specific DNA fragments

Question 2

What is used in PCRs

Answer 2

DNA fragments + DNA polymerase (taq polymerase) +primers + Nucleotides

This cycle is repeated 30-40 times to make lots of copies of DNA fragments

Question 3

What occurs in PCR

Answer 3

Heat at 90 degrees Celsius to separate DNA strands to break hydrogen bonds
Allows primers to bind to DNA fragment template strand
By forming hydrogen bonds between complementary bases
Optimum temperature for DNA polymerase (taq polymerase) (72 degrees Celsius)
Nucleotides align next to complementary exposed bases
DNA polymerase joins the adjacent nucleotides together forming phosphodiester bonds
Repeat cycle many times

Question 4

What are primers

Answer 4

Short, single stranded DNA fragments which are complementary to template of DNA at the edges of the region to be copied .
DNA polymerase binds to primer to start synthesis
It can only add nucleotides onto pre-existing 3’ ends

Question 5

Why are two different primers needed

Answer 5

DNA strands run in anti-parallel, but polymerase can only run in one direction.