6.3.1 DNA Sequencing

Question 1

What is DNA Sequencing?

Answer 1

When an organism’s nucleotide base sequence (genetic material) is identified and recorded

Question 2

What are dideoxynucleotides?

Answer 2

• Used in the chain termination method
• Modified nucleotides
• Pair with nucleotides with complementary bases on template strand during DNA replication

Question 3

What is the setup of the chain termination method (sanger sequencing)?

Answer 3

• 4 test tubes containing DNA to be sequenced
• In form of template strand (single strand)
• DNA Polymerase
• DNA Primers
• Nucleotides ACTG and ACTG (different one in each test tube)
• (ACTG)* are dideoxynucleotides complementary to ACTG
• Incubated to temperature at which DNA polymerase can function

Question 4

How does the chain termination method work?

Answer 4

• Primer binds to start of template strand, forming small double stranded DNA fragment
• DNA polymerase attaches to this small double stranded section and begins DNA replication
• Hydrogen bonds form between complementary bases
• DNA polymerase, at any time, can insert a dideoxynucleotide
• Which terminates replication
• Chains vary in length from one to several hundred nucleotides
• (Each test tube contains 1 type of dideoxynucleotide, so if a tube contains A*, then scientists know that the final nucleotide of each chain in the test tube is A. Lots of chains are produced and are separated later)
• Once incubation period has finished, the developing strand is separated from the template strand
• Which are separated by length by gel electrophoresis

Gel electrophoresis
- 4 wells, one for each of ACTG
- Fragments 1 nucleotide in length travel to end of well, and with the addition of 1 base, each fragment separates 1 band below the last
- So that way scientists can observe which nucleotide base occurs at which point on the strand
- Genome has been sequenced

Question 5

Why is ‘next generation sequencing’ better than the chain termination technique?

Answer 5

• Quicker (millions of DNA molecules sequenced at same time)
• Costs are reduced
• Eg. Nanopore sequencing, High Throughput sequencing