DNA sequencing means working out the order of bases (A, T, C and G) in a piece of DNA. The classic method is Sanger sequencing, also called the chain termination method.The DNA to be sequenced is mixed with:
• a primer
• DNA polymerase
• normal free DNA nucleotides
• a small amount of each of four terminator nucleotides (dideoxynucleotides), each with a different coloured fluorescent label. A terminator nucleotide lacks the -OH group needed to attach the next nucleotide, so when one is added, the new strand stops growing.The DNA is denatured to single strands and the polymerase builds new complementary strands from the primer. At each position there is a chance that a terminator nucleotide is added instead of a normal one. This produces a mixture of fragments of every possible length, each ending in a coloured terminator that shows the last base.The fragments are separated by size using capillary electrophoresis. The shortest fragments move fastest and pass the detector first. A laser detects the colour of each fragment in turn, and a computer converts the order of colours into the base sequence. This gives the sequence of the new strand, which is complementary to the original template.
High-throughput sequencing (next-generation sequencing) methods are now used. Millions of DNA fragments are sequenced at the same time and automated, so whole genomes can be sequenced in hours at a fraction of the original cost. Computers then overlap the short sequences to rebuild the full genome.
• a primer
• DNA polymerase
• normal free DNA nucleotides
• a small amount of each of four terminator nucleotides (dideoxynucleotides), each with a different coloured fluorescent label. A terminator nucleotide lacks the -OH group needed to attach the next nucleotide, so when one is added, the new strand stops growing.The DNA is denatured to single strands and the polymerase builds new complementary strands from the primer. At each position there is a chance that a terminator nucleotide is added instead of a normal one. This produces a mixture of fragments of every possible length, each ending in a coloured terminator that shows the last base.The fragments are separated by size using capillary electrophoresis. The shortest fragments move fastest and pass the detector first. A laser detects the colour of each fragment in turn, and a computer converts the order of colours into the base sequence. This gives the sequence of the new strand, which is complementary to the original template.
High-throughput sequencing (next-generation sequencing) methods are now used. Millions of DNA fragments are sequenced at the same time and automated, so whole genomes can be sequenced in hours at a fraction of the original cost. Computers then overlap the short sequences to rebuild the full genome.