6.1.3: Manipulating genomes

Question 1

Stages of DNA profiling

Answer 1

1) Extract the DNA e.g. from tissue sample (usually need to get more DNA for analysis using PCR)
2) Digest the sample using restriction endonucleases
3) Separate the fragments using gel electrophoresis
4) Visualise the DNA using DNA probes (hybridisation)

Question 2

Why do bacteria contain restriction endonucleases?

Answer 2

To cut up viral DNA & prevent themselves from invading viruses

Question 3

What do restriction endonucleases do?

Answer 3

Cut DNA at specific base sequences –> can produce blunt or sticky ends

Question 4

Main components of gel electrophoresis

Answer 4

• Agarose gel
• Buffer solution to keep the pH constant
• Electrodes placed at each end of the tank

Question 5

Which electrode do DNA fragments move towards and why?

Answer 5

The anode (+ve electrode) because DNA is negatively charged due to phosphate backbone

Question 6

What does the speed of DNA fragment travel depend on?

Answer 6

• The size of the particle; smaller = less friction = quicker
(•sometimes the charge of the particle)

Question 7

Uses of DNA profiling?

Answer 7

• Forensic investigations
• Checking parentage
• Wildlife protection (detecting illegally traded animals)
• Testing food (for source/genetically modified ingredients)

Question 8

What does reverse transcriptase do?

Answer 8

• Used to turn viral RNA into DNA

so that it can be transcribed by the host cell into proteins

Question 9

Why is reverse transcriptase useful e.g. for taking the insulin gene and putting it into bacteria?

Answer 9

• Bacterial DNA does not contain introns, thus bacteria do not have enzymes for splicing
• Proper translation would not occur
• cDNA (product of reverse transcriptase process) des not contain introns

Question 10

What is PCR?

Answer 10

• Polymerase Chain Reaction
• Technique used to amplify a sample of DMA 1000s of times to create a large enough sample for extensive analysis
• Can only amplify short sequences of DNA (few 100 bp long)
• Is in vitro amplification of DNA or gene cloning

Question 11

Uses of PCR

Answer 11

• Paternity tests
• Detecting mutations (comparing different samples of DNA)
• To create enough DNA from a small sample to create a DNA profile
• Molecular biology experiments

Question 12

What is needed for PCR?

Answer 12

• The DNA fragment to be copied
• DNA polymerase (taken from bacteria from hot springs so can withstand high temperature)
• Primers
• Nucleotides to build new DNA with
• Thermocycler: computer-controlled machine that varies temperature precisely for set time periods

Question 13

PCR steps

Answer 13

1) Denature the DNA
⟶ Heat to 95⁰C to separate the two strands
⟶ More C–G = may need higher temp because more H bonds
2) Anneal the primers
⟶ Temp cooled to 55⁰C allowing primers to bind
⟶ Prevents two DNA strands from joining back together
3) Extension of DNA
⟶ 72⁰C = optimum temp for DNA polymerase
⟶ DNA polymerase moves along strand catalysing formation of phosphodiester bonds between adjacent DNA nucleotides
BY THE END THE AMOUNT OF DNA PRESENT HAS DOUBLED.

Question 14

What is a primer?

Answer 14

A short sequence of single stranded DNA that signals to DNA polymerase where to bind.
(DNA polymerase can only bind to double stranded DNA)

Question 15

Pros of in vitro cloning

Answer 15

✔︎ Fast

✔︎ No cells used (cells can die without warning)

Question 16

Pros of in vivo cloning

Answer 16

✔︎ Less risk of contamination of DNA (PCR requires very pure sample otherwise contaminated DNA will be amplified)
✔︎ More accurate (have repair enzymes)
✔︎ Bacteria can go on to produce the protein encoded by the DNA

Question 17

What is DNA sequencing?

Answer 17

• Technique that involves reading the base sequence of DNA

* Based on Sanger sequencing (1975)

Question 18

How does DNA sequencing work?

Answer 18

• Four test tubes labelled A, T, C, G
• Each contains a corresponding modified dideoxy nucleotide that cannot form a phosphodiester bond and so stops further synthesis
• Tubes –> thermal cycler
• DNA polymerase synthesises copies of the DNA sample
• From time to time at random a dideoxy nucleotide will be added to the growing chain and synthesis will then stop
• Range of DNA molecules are synthesised, ranging from v. short to full length, all ending in the labelled nucleotide
• Run contents of tube side by side on electrophoresis gel
• Fragments are sorted by length so sequence can simply be read off from the smallest fragment at the bottom to the largest at the top.

Question 19

What is needed for DNA sequencing?

Answer 19

• DNA sample
• Nucleotides
• Dideoxy nucleotides (at 1% conc)
• DNA polymerase
• Electrophoresis equipment

Question 20

What is cycle sequencing?

Answer 20

• Modified method of Sanger sequencing
• Fluorescent dideoxy nucleotides used
• Polymerisation done in single tube using PCR-like reaction
• Fragments separated using capillary electrophoresis
• Gel read by laser beam, sequence of colours converted to a DNA sequence
• Can read 12,000 bases per minute

Question 21

Genetic engineering steps

Answer 21

1) Isolation of DNA containing the required gene
2) Insertion of DNA into vector
3) Transformation: transfer of DNA into a suitable host
4) Identification: finding host organisms containing the vector and DNA
5) Growth/cloning of successful hosts

Question 22

Process of genetically engineering bacteria to produce human insulin

Answer 22

1) Mature mRNA extracted from β cells from Islets of langerhans from human pancreas
2) Reverse transcriptase –> single stranded cDNA
3) DNA polymerase –> double stranded DNA
4) Restriction endonuclease –> cuts plasmid
5) DNA ligase –> sticks back together; recombinant plasmid formed
6) Transformation = heat shock; bacteria permeability increased, they take up the plasmid
7) Transformed cells grow in medium containing antibiotic; only cells that have taken up the original or recombinant plasmid survive
8) Use of gene marker to identity recombinant plasmid
9) Colonies containing recombinant plasmid will not glow under UV light because insulin gene disrupts GFP gene
10) Large quantities of insulin produced when the bacteria translate the relevant gene

Question 23

Bioinformatics

Answer 23

the development of the software and computing tools needed to analyse and organise raw biological data

Question 24

Electroporation

Answer 24

the use of a very tiny electric current to transfer genetically engineered plasmids into bacteria or to get DNA fragments directly into eukaryotic cells.

Question 25

Genome

Answer 25

all of the genetic material of an organism