(S8) C21 - Recombinant DNA technology

Question 1

(12.3) - Why is the DNA heated to 95 degrees in PCR

Answer 1

• to break the hydrogen bonds between DNA double strand and form separate DNA polynucleotide strands

Question 2

(12.3) - How isolated gene can be replicated by the Polymerase chain reaction

Answer 2

1- DNA sample heated to 95 degrees
2- this breaks the hydrogen bonds between the base pairs causing the strands to separate
3- two template DNA strands with exposed bases
4- cooled to 55 degrees to allow primer to bind using complementary base pairs
5- free floating DNA nucleotides attach
6- temp is then raised to 72 degrees (optimal temp for taq DNA Polymerase)
7- Taq polymerase joins DNA nucleotides together forming phosphodiester bonds
8- Thermocycle is then repeated

Question 3

(12.3) - Why is DNA Polymerase heat stable in PCR

Answer 3

• Enzyme does not denature at 95 degrees

Question 4

(12.3) - why is the reaction mixture cooled 55 degrees in PCR

Answer 4

• allows hydrogen bonds to form during the binding of DNA primers

Question 5

(12.3) - purpose of DNA primers

Answer 5

• start the process of DNA polymerisation and prevent the single stranded DNA strands rehybridising

Question 6

(12.3) - explain the importance of knowing short base sequences either side of a specific DNA fragment

Answer 6

• for complementary DNA primers to form base sequences with each separated strand

Question 7

(12.3) - explain why it is necessary to produce a variety of primers for PCR

Answer 7

• There are different DNA base sequences at either side of the strand
• as a result different complementary primers are required to form hydrogen bonds

Question 8

(12.3) - suggest uses of the PCR

Answer 8

• crime scene
• gene cloning
• tissue sampling

Question 9

(12.3) - explain why the primers used in the PCR in one experiment will not bind to DNA in another experiment

Answer 9

• DNA primers has specific base sequence
• Base sequence is only complementary and form hydrogen bonds to/with specific DNA fragment

Question 10

(12.3) - Tests for use in criminal cases often take longer due to the DNA sample being very small and contaminated. explain these factors

Answer 10

• very small: multiple PCR cycles are required
• Contaminated: other DNA present, need to identify what DNA is required

Question 11

(12.3) - ways in which the PCR differs from transcription

Answer 11

• Transcription uses RNA polymerase/PCR uses taq DNA polymerase
• Transcription uses RNA nucleotide (uracil)/ PCR uses DNA nucleotide
• Transcription uses one DNA strand as template/ PCR uses both
• Transcription promoter region and start or stop codons/ PCR doesn’t
• Transcription uses RNA polymerase to split two strands/ PCR uses heat (95 degrees)

Question 12

(12.3) - how many DNA molecules will have been produced from 1 fragment of DNA after 6 complete cycles

Answer 12

(2x1)squared by 6