Manipultion of DNA

Question 1

How does recombinant DNA technology work?

Answer 1

It is gene cloning that requires isolating sections of DNA and cloning them to make multiple copies

Question 2

In recombinant DNA technology small pieces of DNA is required.

What enzyme is used to achieve this?

How does they work?

Answer 2

Restriction enzymes

They recognise specific nucleotide sequences and act as molecular scissors to cut the DNA into smaller sections

Question 3

What is the difference between blunt or sticky ends?

Answer 3

When restrictive enzymes cut DNA they can either leave both strands flush (blunt) or angled (sticky)

Question 4

Are blunt or sticky ends best for cloning?

Answer 4

Sticky

Question 5

How can the newly cut DNA fragments be cloned?

What molecule helps with this process?

Answer 5

They are inserted into plasmids

Question 6

What is a plasmid?

Answer 6

A small circular strand of DNA found in bacteria which can replicate independently of chromosomes

Question 7

How does DNA fit into a plasmid?

Answer 7

The same restriction enzymes which cut the DNA also cut the plasmid, allowing a complementary fit to form

Question 8

What is the complex called that consists of a DNA fragment and a cut plasmid?

Answer 8

A recombinant DNA molecule

Question 9

Which enzyme joins the DNA to the cut plasmid?

Answer 9

DNA ligase

Question 10

How is a recombinant DNA molecule cloned?

Answer 10

It is taken up by bacteria which use the origin of replication to produce 100-200 copies

Question 11

What type of DNA is used for the recombinant DNA process for eukaryotic genes?

Answer 11

Mature mRNA as there is not process in bacteria to splice exons and remove introns

The mature mRNA is then converted back to DNA

Question 12

What is the name for DNA which has been produced from mature mRNA?

Answer 12

Complimentary DNA

cDNA

Question 13

Which enzyme is responsible for producing cDNA from mature mRNA?

Answer 13

Reverse transcriptase

Question 14

What is different about cDNA for regular DNA?

Answer 14

It is exons only, as the introns have been removed during the mature mRNA formation

Question 15

What is the difference between deoxynucleotides and dideoxynucleotides?

Answer 15

Deoxynucleotides have an OH at carbon 3 but dideoxynucleotides jut have a H

Question 16

What are ddNTP used for?

How does this process work?

Answer 16

For gene sequencing to identify the sequence of base pairs, as the ddNTP interrupt the DNA polymerase enzyme from extending the sequence, therefore where the sequence ends must be the base of the ddNTP in the mix

E.g. ddATP (adenosine)

Question 17

How is automated DNA sequencing one step above dideoxynucleotide DNA sequencing?

Answer 17

It uses all x4 ddNTP’s at once and they are each marked with a fluorescent marker

Question 18

What is PCR?

What does it do?

Answer 18

Polymerase chain reaction

It is another way of making clones of DNA fragments

Question 19

How does PCR work?

What are it’s X3 steps?

Can you name some primers?

How long does each step take?

What does the temperature need to be for each step?

Answer 19

It uses primers to recognise the sequences of DNA at the ends of small DNA fragments

The steps are:

1) denaturation
= slitting the DNA into single strands at a heat of 95 degrees
= 30 seconds

2) primer annealing
= reducing the temperature to around 45-68 degrees to allow primers (TAQ or PFU) to hybridise
= 30 seconds

3) primer extension
= temperature increased to 72 degrees
= TAQ polymerase extends the primers to synthesise DNA
= 60 seconds