Molecular biology 3 - recombinant DNA technology

Question 1

What is recombinant DNA?

Answer 1

Joining together of DNA molecules from different organisms (which is inserted into a host)

Question 2

Applications of recombinant DNA technology

Answer 2

Vaccine production, protein therapies, pharming, gene therapy, transgenic animals

Question 3

Examples of protein therapies produced using recombinant DNA technology

Answer 3

human insulin, human growth hormone, interferon (cytokines)

Question 4

Example of pharming

Answer 4

Transgenic sheep producing blood clotting factors in their milk to treat haemophilia

Question 5

How can gene therapy be used to treat/prevent disease?

Answer 5

By replacing the mutated gene with a healthy one or adding a new (dominant) gene into genome.

Question 6

What are transgenic animals?

Answer 6

animals that possess an integrated gene or DNA sequence in their genome, which can be passed onto offspring

Question 7

How can transgenic animals benefit farming?

Answer 7

Improve the reproductive performance, increase growth rate, improve carcass composition, improve milk production, increase disease resistance

Question 8

What are the 2 methods used to isolate and amplify a gene?

Answer 8

gene cloning (in vivo) and PCR (in vitro)

Question 9

Outline of gene cloning

Answer 9

1. gene is isolated by restriction enzyme
2. Gene is separated from other fragments by gel electrophoresis
3. Plasmid cut by same restriction enzyme
4. Sticky ends of gene and plasmid anneal by complementary base pairing
5. annealed ends covalently joined by DNA ligase
6. plasmid inserted into bacterium host
7. bacteria grows into colony
8. cloned cells are lysed
9. plasmids isolated by centrifugation
10. gene reisolated using restriction enzyme

Question 10

What are restriction enzymes?

Answer 10

Enzymes that cut double-stranded DNA at specific DNA sequences (recognition sites)

Question 11

Features of recognition sites (where restriction enzyme cuts)

Answer 11

4-6 bp long and palindromic

Question 12

Name of staggered cut made by most restriction enzymes

Answer 12

sticky ends (some leave blunt ends)

Question 13

After the sticky ends anneal, how are the fragments joined by covalent bonding?

Answer 13

Using DNA ligase

Question 14

5 general procedures required for cloning DNA

Answer 14

1. isolating DNA to be cloned
2. selecting a cloning vector e.g. plasmid
3. covalent joining of DNA fragments by DNA ligase
4. moving recombinant DNA into host
5. identifying hosts that contain recombinant DNA

Question 15

What is a vector?

Answer 15

a carrier or delivery agent

Question 16

What is a cloning vector?

Answer 16

small molecule of DNA capable of autonomous replication

Question 17

How are host cells containing the recombinant DNA identified?

Answer 17

The plasmid contains 2 antibiotic resistance genes (ampicillin and tetracycline resistant genes). The foreign DNA is inserted into the tetracycline gene so the bacteria with the recombinant plasmid should be resistance to ampicillin but sensitive to tetracycline. The bacteria are grown on plates containing ampicillin to select for bacteria that have taken up the plasmid. The colonies are then transferred into identical position on two plates: one with ampicillin and tetracycline and one with ampicillin. The colonies that grow only on the ampicillin plate contain the recombinant plasmid.

Question 18

What does the Polymerase Chain Reaction do?

Answer 18

Amplifies specific DNA sequence between two known flanking sequences (used to prepare primers)

Question 19

What are primers?

Answer 19

short, single stranded DNA molecules complementary to the ends of a defined sequence of DNA template

Question 20

Uses of PCR

Answer 20

used to identify microbial DNA in clinical samples (using primer), amplifies DNA in forensic samples etc

Question 21

What is required for PCR?

Answer 21

The double stranded DNA to be amplified (template), primers, dNTP, Taq DNA polymerase

Question 22

3 stages of a PCR cycle

Answer 22

1. Denaturation - separates 2 strands of template DNA (94C)
2. Annealing - primers bind to complementary sequences on template DNA (50-60C)
3. Extension - primers extended by Taq DNA polymerase (72C)
   (repeated 20-40 times)

Question 23

After n cycles, how many copies of the original template DNA is produced?

Answer 23

2^n (doubles after each cycle as each new strand becomes template in next cycle)

Question 24

What are the base sequences at the ends of the PCR product?

Answer 24

The primer sequences

Question 25

Advantages of PCR

Answer 25

1. fast
2. primers are very specific so only target DNA is replicated
3. only needs one template DNA molecule (used in forensics)

Question 26

Steps of gel electrophoresis

Answer 26

1. DNA fragments placed in well in agarose / polyacrylamide gel
2. current applied
3. DNA (-ve) moves towards anode (+)
4. Smaller DNA fragments move further
5. Autoradiography / fluorescent dye used to visualise DNA

Question 27

Example of stain used to visualise DNA in gel electrophoresis

Answer 27

ethidium bromide (illuminated with UV irradiation to become fluorescent)

Question 28

How is ethidium bromide used to detect DNA?

Answer 28

Ethidium bromide intercalates between planar rings of DNA double helix. Fluoresces under UV irradiation.

Question 29

What is DNA sequencing?

Answer 29

process used to determine order of bases in DNA (gene or genome)

Question 30

Alternative name for Sanger sequencing

Answer 30

Dideoxynucleotide chain termination method

Question 31

What is required for Sanger sequencing?

Answer 31

Single-stranded DNA template, primer, dNTP, ddNTP, DNA polymerase, label (e.g. primer of ddNTP)

Question 32

Function of DNA polymerase

Answer 32

Catalyses DNA strand synthesis

Question 33

How do dideoxynucleotides (ddNTP) cause chain termination?

Answer 33

3’C has a H instead of an OH group so a phosphodiester bond cannot form to the next nucleotide.

Question 34

Why are ddNTP added at a much lower concentration than dNTP?

Answer 34

ratio of 1:10 - 1:300 (depends on number of bases in fragment) allows sufficient strand elongation for analysis

Question 35

Which end of the DNA does the primer bind to?

Answer 35

3’ end

Question 36

Procedure of Sanger DNA sequencing

Answer 36

1. DNA mixed with primer
2. primer binds to 3’ end of DNA
3. DNA-primer mixture divided into 4 reaction tube, each containing one of ddATP, ddGTP, ddCTP, ddTTP
4. Chain synthesis catalysed by DNA polymerase
5. Gel electrophoresis
6. DNA bands detected by autoradiography or laser
7. DNA sequence deduced

Question 37

How is the DNA sequence deduced using Sanger sequencing?

Answer 37

Each lane in gel electrophoresis consists of a different ddNTP. Dark bands indicate where chain termination has occurred and therefore the location of the specific base. DNA sequence is read from bottom-up across all 4 lanes. DNA sequence of the original strand is complementary to the one inferred from the gel.

Question 38

Name of an automated Sanger throughput sequencing instead of gel-based

Answer 38

dye terminator sequencing (DNA fragments with dye terminators pass through the capillary tube and the colour is detected by a laser)

Question 39

Comparison of gel based vs automated Sanger sequencing throughput

Answer 39

Automated can sequence more base pairs (750-1000bp) and more samples (96) per run than gel-based (250-500bp and up to 8 samples)

Question 40

Name of technology that can sequence millions of genes at once

Answer 40

high throughput / next generation sequencing (NGS) - requires bioinformatics analysis

Question 41

Advantage of using high throughput / next generation sequencing

Answer 41

Cheap and rapid