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

Question 6

Why is the production of sticky ends important?

Answer 6

By using the same restirction endonuclease it allows the combining of any DNA of one organisms with that of another organism

Question 7

Describe the process of in vivo cloning

Answer 7

• Isolate the gene of interest using restriction endonuclease and bind a promoter region
• This allows RNA polymerase to bind, then cut the plasmid using the same restriction endonuclease
• Insert the gene of interest into the plasmid and join them together using DNA ligase
• The plasmids can then be reintrodcuced to the bacterial cells
• Identify those that have taken up plasmid via ampicillin and those with the gene of interest using tetracycline

Question 8

Describe the process of in vitro cloning

PCR (polymerase chain reaction)

Answer 8

• Heat DNA to 95 degrees to break hydorgen bonds holding DNA strands together to seperate them
• Cool to 55 degrees to allow the annealing of primers to the DNA strand
• Heat to 72 degrees to allow DNA polymerase to join and attach the free DNA nucleotides

This process required DNA poylmerase, free DNA nucleotides and primers

Question 9

Three points

What are the advantages of in vivo cloning?

Answer 9

• It involves almost no risk of contamination
• It is very accurate
• It is able to cut out specific genes

Question 10

Two points

What are the advantages of in vitro cloning?

Answer 10

• Extremely rapid process
• It does not require living cells

Question 11

Three points

What are th possible benfits of DNA technology?

Answer 11

• Replacing defective genes which might be able to cure genetic disorders e.g cystic fibrosis
• Genetically modified crops e.g drought resistance
• Production of insulin for people with Type 1 diabetes

Question 12

What is a DNA probe and the two different types?

Answer 12

Short, single-stranded length of DNA that has complementary bases to a specific base sequence
There are radioactively labelled probes and fluorscently labelled probes