Genetic engineering 2

Question 1

What is the purpose of DNA sequencing?

Answer 1

• To PREDICT the function of a DNA sequence
• To IDENTIFY exact differences between normal and mutated genes
• to CONFIRM sequences of engineered DNA

Question 2

To infer DNA sequence from DNA size what can you do?

Answer 2

1) SYNTHESIS the DNA one base at a time
or
2) DEGRADE the DNA one base at a time

Question 3

What is required for DNA sequencing?

Answer 3

• A primer: 15-25bp ssDNA
  (must be synthesised, start with a known sequence)
• DNA polymerase
• Terminator nucleotides (with ratio normal to t 100:1)

Question 4

What are terminator nucleotides?

Answer 4

• synthetic ddNTP (DIdeoxynucleoside triphosphate)
• Lacks OH group at C3
• Lacks phosphodiester bond
• Can be incorporated into the DNA strand being synthesised and prevent subsequent addition of further nucleotides

Question 5

What is the difference between dNTP and ddNTP?

Answer 5

dNTP is a normal deoxynucleoside trisosphosphate (OH group at C3)

ddNTP is dideoxynucleoside T (no OH at C3)

Question 6

What is the ration of dNTP : ddNTP?

Answer 6

100:1

Question 7

What is the process of the chain termination method?

Answer 7

1) Spike different tubes with different terminators
and ifferent chain lengths from each tube - indicate positions of corresponding base terminator nucleotide

Or

1) Put all terminator nucleotides in the same tube but label them with a different colour fluorescent dye
2) Terminator nucleotide added to the 3’ end of the synthesised strand is complimentary to the corresponding position on the template strand
3) Use electrophoresis to separate the fragments depending upon size

Question 8

What gel is used in the chain termination method and why?

Answer 8

• Polyacrylamide gel as it is best used for smaller fragments, differing in 1 bp in length
• Allowing high resolution (1 bands don’t appear as 1)

Question 9

What does RNA sequencing allow?

Answer 9

Determination og aetiology of a disease that occurs due to differing amounts of an expressed gene

Question 10

What do restriction enzymes allow?

Answer 10

Analysis of DNA

DNA engineering

Question 11

What are restriction enzymes?

Answer 11

• Precise cutters of DNA which recognise palindromic sequences
• Cleave double stranded DNA (not RNA or single stranded)
• Each RE recognises and cleaves a specific site

Question 12

Where are restriction enzymes originally isolated from and how are they named?

Answer 12

Bacteria, where they are thought to protect the bacterium from bacteriophages

Names derived from the source of bacteria (eg. EcoRI from E.coli)

Question 13

How do bacteria protect their own palindromic sequences from restriction enzymes?

Answer 13

Methylation of restriction sites, masking them

Question 14

What ends do restiction enzymes make when they cleave DNA?

Answer 14

Sticky ends
- Overhangs

Or

Blunt ends
- Cut DNA at same position on both strands

Question 15

What do the ‘sticky ends’ allow?

Answer 15

Formation of recombinant DNA

- Compatible cohesive ends can ligate to form a new ddDNA molecule

Question 16

What is recombinant DNA?

Answer 16

DNA that has been formed artificially by combining constituents from DIFFERENT ORGANISMS

Question 17

What is the structure of the restriction sites of restriction enzymes?

Answer 17

Short (4-8bp)

Palindromic

Question 18

What does the ‘frequency of restiction sites’ assume?

Answer 18

Assumes that DNA base sequences are random - this is not the case

• Some sequences are overpresented (eg. promoter sequences)