Molecular Techniques

Question 1

Outline gene cloning

Answer 1

1- isolate relevant gene using restriction enzymes to make sticky ends
2- insert gene into plasmid vector to form recombinant DNA molecule
3- insert into host cell (bacteria - rapid replication)
4- isolate clone of DNA

Question 2

Reasons for gene cloning

Answer 2

• to make useful proteins e.g. insulin
• research - find out what genes do
• genetic screening
• gene therapy > replace mutant copy

Question 3

Outline restriction analysis

Answer 3

1- restriction enzymes cleave specific DNA sequences into fragments at restriction site
2- bacteria produce endonuclease as an immune response

Question 4

What is the point of sticky ends?

Answer 4

Only restick pieces of DNA with corresponding sticky ends

Question 5

Reasons for restriction analysis

Answer 5

• investigate size of DNA fragments
• investigate mutations
• investigate DNA variation
• DNA cloning

Question 6

Outline the process of PCR testing

Answer 6

• uses thermostable DNA polymerase Taq
• a pair of primers are made which define the sequence to be copied
• cycle made to replicate sequence exponentially:
  ->95° - DNA denatures + separates strands
  -<60° - annealing
• elongation: Taq polymerase adds nucleotides

Question 7

Uses of PCR

Answer 7

• amplifies DNA
• investigate single base mutations e.g sickle cell
• investigate deletion/insertions
• investigate variation

Question 8

Outline DNA gel electrophoresis

Answer 8

• separates fragments by size
• DNA placed into negatively charged wells
• DNA is negatively charged so moves from negative wells to positive side
• smaller sized fragments can travel further + faster
• visualised under UV light + compared to known DNA

Question 9

Examples of techniques used for analysis at nucleotide level

Answer 9

DNA sequencing
DNA gel electrophoresis
PCR

Question 10

Two types of antibodies

Answer 10

Polyclonal
Monoclonal

Question 11

Describe polyclonal antibodies

Answer 11

• produced by many B lymphocytes
• multiple different antibodies
• specific to one antigen
• many epitopes

Question 12

Describe monoclonal antibodies

Answer 12

• produced from one B lymphocytes
• 1 identical antibodies
• specific to 1 antigen
• 1 epitope

Question 13

Outline western blotting

Answer 13

• detects presence of protein in a tissue
• primary antibody binds to target protein
• enzyme linked secondary antibody binds to complex
• substrate added + colour produced

Question 14

Outline ELISA

Answer 14

• enzyme linked immunosorbent assay
• detects proteins conc. in mixture by using antibodies to bind to protein
  .
• antigen coated well
• specific antibody binds to antigen
• enzyme linked antibody binds to complex
• substrate added to change colour
• rate of colour formation is proportional to amount of specific antibody