Free DNA Sequencing OCR A Level Biology revision notes – covering specification point 6.1.3(a).
DNA sequencing is the process by which the nucleotide base sequence of a gene* is determined so that it can be read.
*Or any other section of DNA.
Types of DNA Sequencing
The principles of DNA sequencing are demonstrated in Sanger sequencing and in high-throughput sequencing:
- Sanger sequencing was effective, but slow and labour-intensive.
- High-throughput sequencing refers to many modern approaches which improve upon Sanger sequencing with chemical and technical methods that allow for DNA to be sequenced faster and more cheaply with the use of computers.
Sanger Sequencing
Sanger sequencing utilises many single-stranded DNA copies of the section of DNA being sequenced.
The single-stranded DNA copies are used as templates for DNA polymerase to carry out DNA replication (turning the single-strands into double-strands), but the experiment is set up so that DNA replication is terminated randomly, resulting in double-stranded DNA molecules of varying lengths.
Gel electrophoresis is used to separate the DNA fragments by length after they are produced.
The terminal (end) nucleotide on the DNA fragments is identified by its marker, and its relative position to the other fragment’s markers is noted to work out the sequence of nucleotide bases in the DNA fragment.
The process of Sanger sequencing can be summarised as:
- The DNA being sequenced is amplified by PCR*, and heated to 94–96 °C to break the hydrogen bonds between the DNA strands to produce single-stranded DNA**.
- A primer is added to the DNA that binds to the template strands, which allows DNA polymerase to bind and add nucleotides to the 3′ end of the strand being synthesised.
- The DNA is divided between 4 separate reaction vessels containing a high proportion of unmodified nucleotides and a small proportion of one of the 4 types of modified nucleotides in each.
- DNA polymerase is added, synthesising many complementary strands using the free activated nucleotides in the 5′ → 3′ direction.
- Whenever a modified nucleotide is added onto a DNA strand, DNA polymerase stops DNA synthesis.
- Many fragments of different lengths are synthesised in each reaction vessel, each ending with the specific modified nucleotide in its respective reaction vessel.
- Once no more reactions are occurring, the fragments are separated by gel electrophoresis.
- The base sequence is determined by identifying the fluorescent/radioactive label of each fragment from smallest to largest.
*The polymerase chain reaction is studied in greater detail later on.
**Breaking the hydrogen bonds between complementary bases with heat is known as ‘denaturing’ the DNA.
High-Throughput Sequencing
High-throughput sequencing refers to a range of modern DNA sequencing technologies that allow DNA to be sequenced faster, more cheaply, and on a much larger scale than Sanger sequencing.
It is important to note that you are not expected to know the details of the techniques relating to high-throughput sequencing (such as pyrosequencing). However, you should be aware of them as examples of techniques that have improved upon Sanger sequencing.
The table below outlines the key developments that have increased the speed and reduced the cost of DNA sequencing:
| Development | Improvement |
|---|---|
| Fluorescent labelling | Eliminating manual reading of DNA sequences from gel electrophoresis, enabling automated laser-scanning reading of the DNA sequence. |
| Pyrosequencing | Enabled DNA sequences to be determined in real-time as computers detect the light signals produced as nucleotides are added. |
| Shotgun sequencing | Enabled the simultaneous DNA sequencing of an entire section of DNA, rather than sequencing it from beginning to end. |
| Massive parallel sequencing | Increased DNA sequencing to a scale that made whole genome sequencing fast and affordable. |