RECOMBINANT DNA TECHNOLOGY

Question 1

Explain how complementary DNA is made using reverse transcriptase

Answer 1

1) strand of mRNA from beta cells of human pancreas coding for production of insulin
2) mRNA acts as a template for production of single stranded complementary copy of DNA (cDNA) using reverse transcriptase
3)cDNA isolated by hydrolysis of mRNA using DNA helicase
4) double stranded DNA formed on the template of cDNA using DNA polymerase
5) results in human insulin gene

Question 2

Explain how restriction endonucleases are used to cut DNA into fragments

Answer 2

-Restriction endonucleases are enzymes that can cut DNA at a specific base sequence
-base sequence that recognise is called the recognition site
-the enzyme cuts DNA between the two specific bases (cleavage site)
-this produces sticky ends - staggered pieces of DNA that are complementary to each other

Question 3

Explain what sticky ends are + their importance in recombinant DNA technology

Answer 3

-DNA is cleaved at a restriction site
-restriction endonucleases cuts out the DNA fragment
-staggered cut leaves a ‘sticky end’ several nucleotide bases long
-any piece of DNA cut with same restriction site will have sticky ends that are complementary
-complementary bases of the 2 sticky ends bind
-DNA ligase joins sugar phosphate backbone
-allows us to bind DNA from one organism to another provided the same restriction endonucleases is used