Sequencing Methods (AQA A Level Biology): Revision Note

Sequencing methods

• DNA sequencing is the process of determining the exact order of nucleotide bases (A, T, C, G) in a DNA molecule

• DNA sequencing allows for the base sequence of an organism's genetic material to be identified and recorded

• Sequencing methods are continuously evolving and becoming faster. Advances in technology have allowed scientists to rapidly sequence the genomes of organisms

• Most sequencing methods used are now automated

• The data obtained from sequencing can be entered into computers with specialised programmes that compare the base sequences of different organisms

DNA sequencing

• All methods of DNA sequencing use dideoxyribose nucleotides

• A dideoxyribose molecule is very similar in structure to ribose molecules and deoxyribose molecules

  • It has one less oxygen atom than a deoxyribose molecule and two fewer oxygen atoms than a ribose molecule

• Dideoxyribose can form nucleotides in the same way that ribose and deoxyribose molecules do, by binding to a phosphate molecule and an organic base

• Dideoxyribose nucleotides can pair with deoxyribose nucleotides on the template strand during DNA replication

  • They will pair with nucleotides that have a complementary base

• When DNA polymerase encounters a dideoxyribose nucleotide on the developing strand, it stops replicating. This is the chain-termination technique that is used for DNA sequencing

Automated DNA sequencing

• Automated DNA sequencing makes use of the chain-termination technique

• An automated DNA sequencing machine can read roughly 100 different DNA sequences within 2 hours

• The process is extremely accurate and can detect fragments differing by just one base

• The key steps involved as as follows:

1. Single-stranded DNA is inserted into a vector

2. A primer is added and binds (anneals) to the start of the DNA

3. DNA polymerase is added along with:

   • Normal deoxynucleotides,which allow DNA replication

   • Modified dideoxynucleotides which stop replication when added

     • Each modified dideoxynucleotide is tagged with a unique fluorescent dye

4. DNA replication begins from the primer. Occasionally, a modified dideoxynucleotide is added instead of a normal deoxynucleotide, this causes termination

5. This produces many DNA fragments of different lengths, each ending with a modified dideoxynucleotide

6. Fragments are separated by capillary electrophoresis, which sorts them by size (accuracy to a single nucleotide)

7. A laser excites the fluorescent tags

8. A detector reads the colours (of the dyes) as the fragments pass, identifying the terminal base of each fragment

9. A computer reconstructs the DNA sequence from this data