Unit 1: PCR and DNA sequencing

Question 1

what is PCR (polymerase chain reaction) used for?

Answer 1

PCR can be used to amplify a desired DNA sequence of any origin

Question 2

what are the initial requirement for PCR

Answer 2

a template of DNA
two primers
taq DNA polymerase

Question 3

what are the stages of PCR?

Answer 3

1. components of PCR mixed together - two primers, template of DNA and taq DNA polymerase
2. DNA heated to 95°C to separate double strand of DNA
3. temperature reduced to allow primers to bind to their complementary base pairs on each strand
4. Taq polymerase adds nucleotides to 3’ end and extends new complementary strand - DNA has doubled
5. repeat heating and cooling cycles to multiply DNA

Question 4

what are the stages of thermal cycling?

Answer 4

1. denaturation at 95°C - mixture is heated to 95°C to denature the DNA breaking hydrogen bonds
2. annealing primers at 55°C - cooling allows the primers to anneal forming hydrogen bonds to their complementary base sequence on the single stranded DNA
3. DNA extension at 72°C - optimum temp for Taq polymerase to extend complementary strands in 5’ to 3’ direction

Question 5

what are the uses of PCR?

Answer 5

cloning DNA fragments
detecting a virus in the very early stages of infection (HIV)
forensics

Question 6

what is DNA sequencing?

Answer 6

the determination of the order of the nucleotide bases in a fragment of DNA

Question 7

what is the difference between deoxynucleotide and a dideoxynudleotide?

Answer 7

dideoxynucleotide lacks a hydroxyl group on carbon 3 and so prevents further bond formation so no more extension of the DNA polymer occurs

Question 8

what is another term for dideoxynucleotides?

Answer 8

chain terminator

Question 9

what are the two methods of DNA sequencing?

Answer 9

sanger sequencing method

dye-terminating sequencing method

Question 10

what is the sanger sequencing method?

Answer 10

the primer is radioactively labelled
four PCR incubations are carried out
in each one, a different dideoxynucleotide is added ( ATC or G)
different chain terminators produce different selection of fragments in four incubations
fragments run in four lanes in gel electrophoresis
once the result has been blotted and exposed to photographic film, base sequence can be read

Question 11

what is dye-terminating sequencing?

Answer 11

each four chain-terminating dideoxynucleotides are tagged with a different colour of fluorescent dye
each unique length will have one fluorescent tag, gel electrophoresis separates the, optical reader detects final base

Question 12

what are the stages of Dye-terminating sequencing

Answer 12

stage 1. DNA replication in presence of four dideoxynucleotides and four deoxynucleotides to amplify the DNA fragments
stage 2. each fragment terminated by a fluorescent dye
stage 3. fragments separated by gel electrophoresis
stage 4. detector identifies the coloured dye of each fragment and therefore the sequence of the fragment