Biotechnologies

Question 1

What is gel electrophoresis?

Answer 1

A method of separating pieces of DNA according to their size

Question 2

What are the steps of gel electrophoresis?

Answer 2

1) DNA is loaded into wells at the negative end of a gel bed.

2) An electric current is passed through the gel.

3) The negatively charged DNA moves towards the positive charge at the opposite end of the bed

4) The DNA moves through the gel at different speeds – smaller pieces move faster than larger ones

5) This forms bands representing different sized segments of DNA. Bands may be called a DNA profile or DNA fingerprint.

Question 3

What is the purpose of PCR

Answer 3

To amplify small amounts of DNA

Question 4

What reactants are added to the PCR tube?

Answer 4

Restricted DNA sample
Leading & Lagging strand primer
DNA Polymerase (taq polymerase)
Free deoxyribonucleotides

Question 5

What is the process of PCR?

Answer 5

1 - Denaturing) DNA sample heated to 95C, causes double helix to break into two strands

2 - Annealing) DNA primer anneals to complimentary leading & lagging strands of DNA at temp of 50C-65C

3 - Elongation) Taq polymerase brings in corresponding nucleotides to bind to complimentary nucleotides on the strands of DNA at 72C

4 - Repetition) Steps 1-3 are repeated using a thermocycler

Question 6

What is chain termination sequencing?

Answer 6

Process used to determine the exact sequence of nucleotides in a DNA sequence.

Question 7

What are the reactants in chain termination sequencing?

Answer 7

Restricted DNA sample
Fluorescent dye
Dideonucleotides
Free nucleotides
Leading & lagging strand primer
Taq polymerase

Question 8

What is the process of chain termination sequencing?

Answer 8

1 - DNA preparation) Cell membrane is broken apart (precipitation) and stored in a vector (plasmid)

2 - Sequencing reaction) Reactants added into a PCR tube, allowing fluorescent tags to attach to dideonucleotides. Process of PCR is undergone

3 - Capillary Electrophoresis) Resulting DNA is loaded into capillary tube, process of gel electrophoresis is undergone.
Laser is passed through strands, colours of fluorescent tags are ID’ed, info sent to a computer.

4 - Computer analysis) Info displayed as electropherogram, bases written below each peak.
Overlapping sequence is put together, and used to figure out the original DNA sequence.