Section 8:Recombinant DNA

Question 1

What are the five main stages of DNA technology?

Answer 1

1. Isolation
2. Insertion
3. Transformation
4. Identification
5. Growth/Cloning

Question 2

What are the three methods of producing DNA fragments?

Answer 2

Using reverse transcriptase
Using restriction endonucleases
The ‘gene machine’

Question 3

How does reverse transcriptase produce DNA fragments?

Answer 3

A cell which produces the desired protein is selected~this contains mRNA which is then extracted
The mRNA is used to make complementary DNA (cDNA)
DNA polymerase is used to make the DNA double stranded

Question 4

How does restriction endonucleases produce DNA fragments?

Answer 4

Cuts DNA double strand at a specific base sequence called a recognition site (palindromic)
* Cut between two bases,staright edge=blunt ends formed
* Cut in a staggered fashion each strand has exposed unpaired bases=sticky ends formed

Question 5

How does the gene machine produce DNA fragments?

Answer 5

• Base sequence of gene is discovered from the protein that would be produced
• From this the AA sequence is worked out,mRNA codons are looked up and cDNA bases are worked out
• Gene sequence is fed into a computer to produce oglionucleotides~joined together and replicated using PCR
• PCR also constructs the complementary strand of nucleotides to make the required double stranded gene, this gene is then multiplied many times
• sticky ends the gene is inserted into a bacterial plasmid