Manipulating genomes

Question 1

Polymerase Chain Reaction (PCR)

Answer 1

1.Get double-stranded DNA sample
2.Heat to 95 degrees so hydrogen bonds break causing strands to separate
3.Add primers and reduce temp to 55 degrees so primers can anneal to ends of DNA (needed for replication of strands to occur)
4.Raise temp to 72 degrees (thermophilic bacteria found in hot springs)so DNA can extend using primers using free nucleotides (building up complementary strands of DNA and so producing double-stranded DNA identical to original sequencing)

Question 2

Restriction endonucleases definition

Answer 2

-group of enzymes breaking down bonds within nucleotides
-restriction means target specific sequences of DNA called recognition sites

Question 3

Electrophoresis definition

Answer 3

-a procedure for separating DNA fragments based on size
-key in DNA profiling, but principle also used in DNA sequencing

Question 4

Mechanisms

Answer 4

-‘cleave’ DNA= cut it
-many leaves ‘sticky ends’ (cut in offset fashion)- allowing two sources cut with the same restriction enzyme and complementary sticky ends allow them to anneal
-DNA ligase joins together phosphate-sugar backbone

Question 5

Electrophoresis procedure

Answer 5

1.extraction of DNA from sample
2.digestion-restriction enzymes cut DNA into fragments
3.seperation-fragments using gel electrophoresis
4.seperation-DNA fragments transferred from gel to nylon membrane in Southern binding
5.hybirdisation-DNA probes added to labelled fragments
6.development-membrane with radioactively labelled DNA fragments is placed onto X-ray film
7.development-develop of X-ray film reveals dark bands where radioactive or fluorescent DNA probes have attached