Week 3 Molecular biology techniques 2

Question 1

Expression

Answer 1

The process in which a specific gene is activated to produce a certain protein

Question 2

Vector

Answer 2

A DNA molecule that carries a specific segment into a certain cell, is extrachromosomal and circular

Question 3

Restriction enzyme

Answer 3

Cuts the DNA strands so the gene can be inserted into the vector, recognises a particular sequence of DNA

Question 4

The 3 sections of vectors

Answer 4

Cloning site - DNA is attached
Antibiotic resistant gene - Allows selection of DNA which contains recombinant DNA
Origin of replication - Allows replication

Question 5

The 4 steps of DNA cloning

Answer 5

1. Restriction enzymes cut DNA, the same enzymes cut the vector, both have the same ends so they can be ligated together
2. Transformation - Uses electroporation to create holes in the membrane through which DNA enters
3. Select bacteria with recombinant DNA - If cells replicate, they have a vector
4. Isolating recombinant DNA from the bacteria - Special techniques are used to extract DNA from bacteria

Question 6

Why should we analyse RNA?

Answer 6

To show what genes are being expressed ,shows the change of expression when we change its environment

Question 7

How do we view RNA?

Answer 7

Unstable, use verse transcriptase to convert into cDNA, can be analysed using PCR

Question 8

Western Blotting

Answer 8

To assess protein function in cells

Question 9

Western blotting steps

Answer 9

1. Protein extracted from cells
2. Polycerylamide gel to separate proteins according to size
3. Proteins transferred from gel to membrane
4. Membrane incubated with antibodies which binds to protein we want to detect
5. 2nd antibody binds to 1st
6. Use enzyme horseradish peroxidase which attaches to antibody, causes light emission

7.Light detected by photographic film

Question 10

Why use western blotting?

Answer 10

Detects antibodies which show if the disease is present, detects proteins in body fluids