module 6 processes

Question 1

What do terminator bases do?

Answer 1

• stop DNA synthesis

Question 2

What are the ingredients for DNA sequencing?

Answer 2

• DNA polymerase, primer, excess of nucleotides, terminator bases (fluorescent ddNTPs) and DNA to be sequenced.

Question 3

Why do ddNTPs stop DNA synth?

Answer 3

• lack a 3’ OH group

Question 4

describe the process of DNA sequencing

Answer 4

• DNA sample heated to 95C (denaturation)
• Cool to 50C (primers anneal)
• Heat to 60C. (DNA polymerase can start adding complementary bases)
• Incorporation of a ddNTP means synthesis cannot continue
• Cycling continues until all possible strands/fragments are produced
• Gel electrophoresis –> southern blotting on nylon membrane; UV light. Or capillary electrophoresis which separates by mass and uses colour-detecting lasers.
• Both show ddNTPs, meaning the final base of each fragment can be read and thus the base sequence of the complementary strand is revealed.

Question 5

describe the process of genetic fingerprinting

Answer 5

• collect a sample containing some DNA
• extracted and PCR done
• Digested with specific restriction endonucleases; cut at specific recognition sites
• separation with gel electrophoresis with alkaline buffer (this forms ssDNA)
• DNA hybridises with fluorescent/ radioactive probes
• transferred to nylon membrane and exposed to UV or X-rays

Question 6

How can electrophoresis be used for proteins?

Answer 6

• Proteins must be denatured to remove 3D shape
• Proteins can be positive, negative or neutral. SDS is added to make them all negative

Question 7

Describe the ingredients of PCR

Answer 7

• thermocycler
• DNA fragment to be amplified, taq polymerase, primers, DNA nucleotides

Question 8

Describe the process of PCR

Answer 8

• Heated to 95C to break hydrogen bonds; ssDNA
• Primers anneal at 55C
• DNA polymerase attaches new complementary free nucleotides and makes a new strand complementary to each template. 72C

Question 9

What are 3 temperatures used in PCR

Answer 9

95C, 55C, 72C

Question 10

Advantages of PCR?

Answer 10

• automated: more efficient
• rapid: 100 billion copies of DNA can be made within hours
• doesn’t require living cells

Question 11

Describe the process of genetic engineering

Answer 11

• isolating the gene (mRNA reverse transcription, new synthesis or same restriction endonuclease)
• insertion of gene into open plasmid with marker gene. Same endonuclease used so sticky ends are complementary. Ligase catalyses formation of phosphodiester bonds between them
• transformation: increase cell membrane permeability. Calcium ion, heat shock. Electroporation. High concentrations. Enters cytoplasm.
• identification using fluorescent (or other) marker gene.
• Cloning/growth

Question 12

What issues might occur during G.E of bacteria?

Answer 12

• recombinant plasmid doesn’t get inside cell.
• plasmid re-joins before the DNA fragment entered.
• DNA fragment sticks to itself (mini plasmid shape), rather than inserting into the plasmid.

Question 13

Genetic engineering of plants

Answer 13

1. Cut a leaf. Sterilise.
2. Expose the leaf to a bacterial suspension containing a Ti plasmid (from agrobacterium tumefaciens) with genes for desired trait and antibiotic resistance.
3. Expose leaf to an antibiotic to kill cells which lack new genes. Callus forms from gene-altered cells.
4. Allow callus to sprout shoots and roots.
5. Transfer to the soil to grow.

Question 14

How can plants be genetically engineered using electrofusion?

Answer 14

• Cell walls must first be removed by cellulases.
• Cells of different types can be fused, using electricity, to form a polypoid, transgenic cell.
• Hormones used to grow new cells walls, callus forms.

Question 15

uses of DNA profiling

Answer 15

crime scenes
paternity testing
evolutionary relationships
disease risk
disaster victims
breeding programmes
classification