Sequencing technology, which determines the base sequence of DNA, is widely applied in genomic medicine, evolutionary research, and criminal investigations. This section provides an overview from the classical Sanger method to next-generation sequencers.
Basic Knowledge
Sanger method (dideoxy method): Developed by Frederick Sanger in 1977. He received his second Nobel Prize in Chemistry in 1980.
Principle:
① A primer is annealed to template DNA, and DNA is synthesized by DNA polymerase.
② In addition to ordinary deoxynucleotides (dNTPs), a small amount of dideoxynucleotides (ddNTPs) is mixed in. Because ddNTPs lack 3'-OH, chain extension stops when one is incorporated.
③ Fragments terminated with A, T, G, and C are separated by electrophoresis according to length, and the base sequence is read.
Today, fluorescently labeled ddNTPs are used, making automatic decoding possible with a single capillary electrophoresis run.
Next-generation sequencing (NGS): A method that reads many short fragments in parallel (short-read sequencing). It can decode the human genome (3 billion base pairs) in several days for tens of thousands of yen. The Human Genome Project (1990-2003) achieved the first complete genome sequence through international cooperation.
Answer: In ddATP, the -OH at the 3' position is replaced by H (it lacks 3'-OH), so it cannot form a phosphodiester bond with the next nucleotide, stopping chain extension. The greatest difference from next-generation sequencing is the degree of parallel processing: the Sanger method is a sequential method that reads one sequence per reaction, whereas NGS reads millions to billions of fragments simultaneously in parallel.
- Sanger method = stop chain extension with ddNTPs and read the sequence from fragment lengths
- ddNTPs lack 3'-OH → cannot form phosphodiester bonds
- Sanger = two Nobel Prizes in Chemistry (1958 and 1980)
- Next-generation sequencing = massively parallel sequencing
- Human Genome Project = 1990-2003; decoded 3 billion base pairs
- Today, one person's genome can be decoded in several days for tens of thousands of yen
Practice
- Explain why dideoxynucleotides (ddNTPs) are used in the Sanger method.
- State the principle by which DNA fragments are separated during electrophoresis.
- Give two reasons why next-generation sequencing has revolutionized genomic medicine.