DNA Technology Part 4 - (Week 10)

Question 1

What is DNA sequencing?

Answer 1

DNA sequencing is the process of determining the sequence of nucleotides in a piece of DNA

Question 2

What biological concept is used in DNA sequencing?

Answer 2

The concept of DNA replication

Question 3

Name the two major types of DNA sequencing methods

Answer 3

• Sanger sequencing (chain-termination/dideoxy sequencing)

- Next generation sequencing

Question 4

What does Sanger sequencing require?

Answer 4

• Many identical copies of the DNA to be sequenced
• Oligonucleotide primer complementary to a short stretch of DNA
• DNA polymerase
• Deoxynucleotides (dNTPs)
• Dideoxynucleotides

Question 5

What are dideoxynucleotides?

Answer 5

Chain terminating nucleotides

Question 6

Describe the 7 steps of (Sanger) DNA sequencing

Answer 6

1. DNA mixed w/ primer and an excess of normal nucleotides & terminator bases w/ fluorescent markers attached
2. Mixture placed in thermal cycler.
   96 deg. DNA strands separate.
   50 deg. primers anneal to the strand
   60 deg. polymerase builds new strands
3. Whenever a terminator base is added chain stops (bases in low amounts added at random) to produce fragment of diff. lengths
4. Fragments are separated according to their length by capillary sequencing (like gel electrophoresis)
5. Fluorescent markers are used to identify final bases & lasers to detect diff. colours
6. The order of bases in the capillary tubes shows the sequence of the complimentary strand
7. The info is fed into a computer & the genome can be determined

Question 7

What will the chromatogram look like after Sanger DNA sequencing has occurred?

Answer 7

• Dideoxyribonucleotides will have different fluorescent tags
• The chromatogram will show order of the nucleotides (from shortest to longest)
• Peak heights represent diff. nucleotide bases

Question 8

What can Sanger DNA sequencing be used for?

Answer 8

It allows for comparisons within a species or among separate species

Question 9

Give a brief description of the next-generation sequencing method

Answer 9

• It uses a single template strand that is immobilised & amplified to produce an enormous number of identical fragments
• These fragments are sequenced in parallel
  (It is a type of high speed, ‘high-throughput’ technology)

Question 10

Name at least one advantage of next-generation sequencing

Answer 10

• High speed
• Can produce enormous volume of sequences
• Currently used for whole genome sequencing, research etc.

Question 11

What is a possible reason that Sanger DNA sequencing is a better method than next-generation sequencing?

Answer 11

Although it is more expensive, Sanger sequencing is more precise & can predetermine the size of relatively longer stretches of DNA

Question 12

What is gene therapy?

Answer 12

The alteration of an afflicted individual’s genes in order to replace a defective allele with a normal allele

Question 13

How can gene therapy be done in order to have a long-lasting effect?

Answer 13

It should be done in dividing cells that are renewing the cell population in certain tissues or generation the whole body (gametes)

Question 14

Give an example of how a gene can be delivered into the body in gene therapy

Answer 14

Through the use of vector (virus, retrovirus etc.) - vectors are used for delivery of genes into specific types of cells e.g. bone marrow

Question 15

What is meant by ‘in vivo’ gene therapy?

Answer 15

When the therapeutic agent is put directly into the body

Question 16

What is meant by ‘ex vivo gene therapy?

Answer 16

When the relevant cells are isolated & replaced/modified to put back into the patient

Question 17

What can gene therapy be used for?

Answer 17

It has great potential in treating disorders traceable to a single defective gene; potentially for customised medicine