DNA sequencing

DNA is found in almost every cell of every living organism.

DNA sequencing is the process used to determine the exact sequence of the nucleotides in a strand of DNA. The order of bases varies between organisms and codes for their unique characteristics. DNA sequencing is used to determine the nucleotide sequence of specific genes, larger genetic regions, whole chromosomes or the entire genome of an organism.

DNA sequencing has opened up new possibilities for understanding life and has a wide range of applications – from medical diagnosis to forensics and from biosystematics to genetically modifying organisms.

One of the most common methods of DNA sequencing is the Sanger or dideoxy technique.

Starting off

The DNA to be sequenced must first be broken into smaller pieces and amplified. This is the process of creating multiple copies and is generally done using a process called PCR.

Next, heat is used to separate the double-stranded DNA molecules into single strands. The solution also contains:

• an enzyme called DNA polymerase
• a high concentration of normal DNA nucleotides (deoxynucleotides, dATP, dGTP, dTTP and dCTP)
• a mixture of terminator dideoxynucleotides (ddATP, ddGTP, ddTTP and ddCTP)
• DNA primers – short pieces of DNA that have been specially selected to bind to the selected DNA and are needed to get the reaction going
• buffer – to maintain the required pH for the reaction.

The next steps: making new DNA

One of the original DNA strands is used as a template for the synthesis of new DNA. The primers anneal to the template strand, and the DNA polymerase enzyme makes a new strand of DNA by creating a complementary sequence of nucleotides drawn from the reaction mixture.

The new DNA strand is made by complementary base pairing with the original DNA template. Because all four ordinary DNA nucleotides are present in large amounts, the chain elongation continues normally – until by chance a dideoxynucleotide (terminator) is added in the place of a normal DNA nucleotide.

The dideoxynucleotides are just like ordinary DNA nucleotides except that one hydroxyl (OH) group has been chemically changed to a hydrogen (H). With normal DNA nucleotides, one nucleotide can be attached to another and so on, forming a chain. The chemical change in a dideoxynucleotide, however, means that no additional nucleotides can be added, hence the name ‘terminator nucleotides’.

The synthesis of new DNA is terminated when one of the dideoxynucleotides is added to the strand. Because there are many more ordinary nucleotides than dideoxynucleotides, some chains will be several hundred nucleotides long before a dideoxynucleotide is added. The end result is a whole lot of new DNA fragments, of varying length, all ending with a dideoxynucleotide.

Separating and analysing the new DNA strands

The newly made DNA fragments can be separated according to their size by gel electrophoresis. A current is passed through a gel, pulling the negatively charged DNA molecules through to the positively charged end of the gel. The pores in the gel mean that the shorter fragments move through more quickly.

The terminator nucleotides at the end of each fragment are labelled with a fluorescent dye. Each of the four types of terminator nucleotides fluoresces a particular colour when passed through an optical scanner at the end of the gel. The particular colour emitted by the terminator nucleotide at the end of the chain is recorded by the scanning device. Each colour represents a different nucleotide (A, T, C or G). In this way, the order of nucleotides in the DNA sequence can be obtained.

De novo sequencing

Sometimes not enough is known about the DNA sequence of interest for it to first be replicated by PCR or for the DNA sequencing primers to be created. ‘De novo’ translates from the Latin meaning ‘from the beginning’.

Where longer DNA sequences are of interest – whole chromosomes or genomes – the DNA is broken into smaller fragments using restriction enzymes or mechanical forces. The fragments can then be cloned into a DNA vector and amplified (replicated) in a bacterial host. These fragments are then purified from individual bacterial colonies and sequenced. Because hundreds of different individual sequences will have been obtained, overlaps between them are used to electronically assemble the sequence of the original DNA strand.

Why is a DNA sequencing useful?

DNA sequencing is a biological technique that many different technologies rely on. For example, it can be used for:

• identifying regions of DNA associated with particular features, including specific diseases or increased susceptibility to specific diseases
• understanding gene expression and how different genes interact
• identification of substances, individuals and species
• other genetic technologies such as gene therapy, genealogy research, plant and animal breeding and genetic modification.