Recombinant DNA Technology

Question 1

What is a mutation?

Answer 1

Permanent alteration in a DNA sequence

Question 2

What are the 3 main causes of mutation?

Answer 2

1. Errors in DNA synthesis that can occur spontaneously at low frequency
2. Chemical mutagens
3. Ionising radiation

Question 3

Which type of mutations can have effects of varying degrees of severity?

Answer 3

Single base mutations

Question 4

What is the simplest type of mutation?

Answer 4

Substitution - substituting one base for another

Question 5

What is a conservative mutation (substitution)?

Answer 5

AA is replaced by one with similar properties - may or may not result in a disease phenotype

Question 6

What is a non-conservative mutation (substitution)?

Answer 6

AA is replaced with one with different properties - much more serious: will result in a mutated protein that could be harmful

Question 7

What is a no mutation (substitution)?

Answer 7

In many cases a change in the third position of the codon does not change an AA - so no mutation will occur

Question 8

What will an insertion mutation cause?

Answer 8

Premature termination

Causes ‘frameshift’ - no longer in correct triplets so AA sequence is completely changed

Question 9

What will a deletion mutation cause?

Answer 9

A different protein to be produced

Causes ‘frameshift’ - no longer in correct triplets so AA sequence is completely changed

Question 10

What will an altered base mutation cause?

Answer 10

Single AA change - can be conservative or non-conservative

Question 11

What disorder is caused by a single-base substitution in the beta-chain of haemoglobin?

Answer 11

Sickle-cell anaemia -this is a non-conservative mutation
More drastic mutations may also occur e.g. deletion or duplication of longer stretches of DNA but even a single base change can lead to a very serious disease

Question 12

What is gene cloning?

Answer 12

Produces a large number of copies of a particular piece of DNA
Process:
- Cut out if genome using restriction enzymes (genetic scissors)
- Gel electrophoresis
- Insert into plasmid
- Modified plasmid inserted into bacterium
- Grow bacteria so multiplies and so more genes are copied
- Gene then re-isolated using the same restriction enzymes

Question 13

What are restriction enzymes?

Answer 13

Enzymes that have been isolated from bacteria

Question 14

What do restriction enzymes do?

Answer 14

• Cut double-stranded DNA at specific DNA sequences
• Sequences are typically 4-6 base pairs in length and ‘palindromic’ i.e. they ready the same in both directions
• Most restriction enzymes make a staggered cut, which allows DNA fragments to re-associate by base pairing
• After re-association, the fragments can be re-joined by DNA ligase

Question 15

What does palindromic mean?

Answer 15

They read the same in both directions

Question 16

What is gel electrophoresis used for?

Answer 16

• Used to separate DNA fragments on the basis of their size
• Samples are applied to a gel immersed in buffer and a current is applied
• Negatively-charged DNA migrates from the negative electrode to the positive electrode
• Larger DNA fragments migrate more slowly than smaller DNA fragments

Question 17

What is the procedure for gene cloning?

Answer 17

• To insert a gene that has been isolated by gel electrophoresis into a plasmid, a restriction enzyme is chosen that cuts on either side of the gene but not in the middle
• The gene is separated from other DNA fragments by gel electrophoresis
• A suitable plasmid is linearized using the same restriction enzyme
• The cut plasmid and gene are mixed, and the sticky ends of the plasmid and gene are allowed to ‘anneal’ (associate by base pairing)
• The annealed ends are covalently joined by using DNA ligase
• The plasmid, now containing the gene of interest, is introduced into the host bacterium
• The bacteria are grown into a colony, using antibiotic resistance genes in the plasmid to select colonies containing plasmids
• Cloned cells are lysed and the plasmids isolated by centrifugation
• Plasmids are cut with the restriction enzyme, releasing the cloned gene

Question 18

What is DNA sequencing used for?

Answer 18

• used to determine base sequence of DNA

- Works out the structure of a gene or an entire genome

Question 19

What is the Sanger sequencing?

Answer 19

• Original - last generation technology nut still widely used for small scale projects
• Synthesis of new DNA strands complimentary to a single-stranded template strand in vitro

Question 20

What are dNTP’s?

Answer 20

Building blocks of DNA - similar to deoxynucleotides

Question 21

What are ddNTP’s?

Answer 21

Modifies nucleotides that terminate DNA strand elongation

Question 22

What is DNA polymerase?

Answer 22

Enzyme that catalyses DNA strand synthesis - incorporates the bases into new strand being made

Question 23

What is a label used for in the Sanger sequencing method?

Answer 23

• Fluorescent or radioactive

- Required to visualise products

Question 24

What is the DNA sequencing procedure?

Answer 24

1. DNA to be sequenced is mixed with primer
2. Primer binds to 3’ end of DNA
3. DNA-primer mixture divided into 4 separate reaction tubes containing:
   - All four dNTP’s
   - One of the four ddNTP’s (A, C, G, T tubes)
   - DNA polymerase
4. Chain synthesis proceeds in each of the four reaction mixtures - so for gene with 500 bases in length will produce 500 products each differing by one base
5. Gel electrophoresis separation of reaction products -> band corresponding to each position of chain termination appears
6. DNA bands detected by autoradiography or by laser in an automated sequencer
7. DNA sequence can be deduced from the pattern of bands in the four lanes
   - A dark band in a lane indicates a DNA fragment that is the result of chain termination after incorporation of a ddNTP
   - The terminal nucleotide base can be identified according to which ddNTP was added in the reaction giving that band
   - The relative positions of the different bands among the four lanes are then used to read (from bottom to top) the DNA sequence which gives the order of sequence of bases

Question 25

What technique is also known as the ‘next generation sequencing’?

Answer 25

The High Throughput sequencing

Question 26

How does the High Throughput sequencing technique work?

Answer 26

• Take the human genome and chop it into many pieces, then by sequencing every DNA fragment at one on a chip
• So can sequence millions of genes and entire genome at once
• This requires substantial bioinformatics analysis