PCR and Electrophoresis

Question 1

What does PCR stand for?

Answer 1

Polymerase chain reaction.

Question 2

What is PCR used for?

Answer 2

To produce large quantities of specific fragments of DNA or RNA from very small quantities.

Question 3

What are primers used in PCR?

Answer 3

Short sequences of single stranded DNA that have bas sequences complementary to the 3’ end of DNA/RNA being copied.

Question 4

What if the function of primers in PCR?

Answer 4

They provide a starting point for DNA polymerase because needs a starting strand onto which to attach nucleotides.

Question 5

Where do primers attach?

Answer 5

Only at a particular position because new nucleotides only attach at 3’ OH end.

Question 6

Why is DNA polymerase used in PCR?

Answer 6

Used to build new DNA or RNA strands.

Question 7

Why is Taq polymerase used instead of DNA polymerase in PCR?

Answer 7

Because it will not denature at high temperatures involved in the first stage of PCR and its optimum temperature is high enough to prevent annealing/joining of DNA strands that have not copied yet.

Question 8

What do free nucleotides do in PCR?

Answer 8

Used in construction of DNA or RNA strands.

Question 9

Why is buffer solution used in PCR?

Answer 9

Provides the optimum pH for the reactions to occur in.

Question 10

Why is the DNA produced exactly the same as the original?

Answer 10

Because complementary bases can only attach to one another and the original strands acts as a template.

Question 11

How can you find out how many molecules are at the end of the PCR cycle?

Answer 11

Do the 2 ^ number of cycles.

Question 12

Why are 2 primers used in PCR?

Answer 12

Because the sequence of bases at the start of each strand are different.

Question 13

Where is Taq polymerase obtained from?

Answer 13

Thermophilic bacteria in hot springs.

Question 14

What is the first stage of PCR?

Answer 14

DENTURATION:
- The double stranded DNA is heated to 95C which breaks the H2 bonds that bond the two DNA strands together.

Question 15

What is the second stage of PCR?

Answer 15

ANNEALING:
- The temperature is decreased to between 50C-60C so the primers can anneal to the ends of the single strands of DNA.
- Reverse and forward primers are used as there are 2 strands so can only attach to the 3’ end so 2 are needed.

Question 16

What is the third stage of PCR?

Answer 16

ELONGATION/EXTENSION:
- The temperature is increased to 72C for at least a minute, as this is the optimum temperature for Taq polymerase to build the complementary strands of DNA to produce the new identical double stranded DNA molecules.

Question 17

Why is electrophoresis used?

Answer 17

To analyse DNA, RNA, and proteins.

Question 18

What is the method of electrophoresis?

Answer 18

1. Create gel plate in tank and wells cut into gel at one end.
2. Submerge gel in electrolyte solution in tank, then insert fragments into wells with pipette.
3. Apply electrical current and attach the negative electrode to end of plate with wells as DNA fragments move to the positive electrode.
4. Smaller piece move faster and further from wells.
5. The fragments are not visible so probes added.

Question 19

How are molecules separated?

Answer 19

According to size and net charge because the positive molecule move to the negative pole but negative molecules move to the positive pole.

Question 20

What charge is DNA?

Answer 20

It is negatively charged so will move to the anode - the positive electrode.

Question 21

How do different sizes molecules move in electrophoresis?

Answer 21

The tiny pores results in the molecules moving in gel at different rate, smaller molecules move quicker than larger ones, different gels have different sized pores so move at different rates.

Question 22

How is DNA separation carried out in electrophoresis?

Answer 22

1. Collected from parts of the body.
2. PCR is used to amplify DNA
3. ‘RE’ to cut DNA into fragments. Different ‘RE’ cut DNA at different base sequences so you must use enzymes that cut close to the VNTR regions (found in non-coding DNA) - contains variable number of DNA sequences.

Question 23

What is protein separations’ method?

Answer 23

Different amino acids determine the charges of proteins, proteins prepared by electrophoresis are denaturing to break disulfide bonds and manipulate them into rod shapes to allow separation by size.

Question 24

What is the use of protein separation?

Answer 24

To show genotypes by separating alleles.

Question 25

Where is Taq polymerase obtained from?

Answer 25

Thermophilic bacteria in hot springs.

Question 26

What happens in each cycle of PCR?

Answer 26

In each cycle, the DNA is doubled, a thermal cycler provides optimum temperature and controls the length of time at each stage.