M11-3 DNA_sequencing_W24.pdf

BIOL 2030 Module 11 11.3:DNA sequencing April 1, 2024 M11-3 1 Molecular toolbox started to fill up! 1970s Type II restriction endonucleases Agarose gel electrophoresis DNA sequencing (Sanger dideoxy method – original version) 1980s Sanger dideoxy method – fluorescent version Polymerase chain reactio...

BIOL 2030 Module 11 11.3:DNA sequencing April 1, 2024 M11-3 1 Molecular toolbox started to fill up! 1970s Type II restriction endonucleases Agarose gel electrophoresis DNA sequencing (Sanger dideoxy method – original version) 1980s Sanger dideoxy method – fluorescent version Polymerase chain reaction Late 1990s – 21st century Illumina DNA sequencing Nanopore DNA sequencing + others… M11-3 2 DNA sequencing: Sanger dideoxy chain terminating method PROFESSOR FRED SANGER (1918-2013) Shown here at the Wellcome Trust Sanger Institute, UK, at circa 90 years of age. Nobel Laureate in chemistry for inventing the process of sequencing of proteins (1958) and DNA (1980). The dideoxy-chain terminating DNA sequencing method was published in 1975. One of two methods of sequencing DNA invented in 1970s. Still used today. Remains gold standard for accuracy and convenience for sequencing small numbers of samples. M11-3 3 Reminder: MINIMUM REQUIREMENTS FOR DNA SYNTHESIS IN VITRO DNA primer 5' 3' 3' DNA polymerase 5' DNA template Most methods of DNA sequencing are based on DNA synthesis Deoxyribonucleoside triphosphates DNA synthesis of the new strand always proceeds in the 5’ to 3’ direction! M11-3 4 FORMATION OF A PHOSPHODIESTER BOND CATALYZED BY DNA POLYMERASE P-P is released from the dNTP during the reaction. H H H H M11-3 5 Dideoxyribonucleoside triphosphates terminate DNA synthesis (a) Deoxyribonucleoside triphosphate (b) Dideoxyribonucleoside triphosphate M11-3 6 Consider what would happen in a DNA extension (synthesis) reaction, in which most of the dGTP is regular dGTP, but a small amount, say 5%, is ddGTP. In that case, most (95%) of the time when ‘G’ is incorporated, it would be a normal dGTP, and strand elongation past that base could continue. BUT, 5% of the time, a ddGTP would be incorporated, and when that happened, there would be no further extension of that particular DNA strand. This would give us DNA daughter strands of varying lengths, the lengths of which are determined by where the ‘G’s occur in the sequence. M11-3 7 Consider what would happen in a DNA extension (synthesis) reaction, in which most of the dGTP is regular dGTP, but a small amount, say 5%, is ddGTP. In that case, most (95%) of the time when ‘G’ is incorporated, it would be a normal dGTP, and strand elongation past that base could continue. BUT, 5% of the time, a ddGTP would be incorporated, and when that happened, there would be no further extension of that particular DNA strand. This would give us DNA daughter strands of varying lengths, the lengths of which are determined by where the ‘G’s occur in the sequence. We could do the same thing for the other bases: ‘Spike’ the DNA polymerization cocktail with small amounts of ddATP, ddCTP, and ddTTP (in addition to the ddGTP). In this case, we would get a subset of DNA elongation products terminating with a ddNTP base at every position in the DNA sequence. BUT – (1) how do we keep track of which bases are terminating which fragments? AND – (2) how do we sort out the different fragments by size? M11-3 8 ANSWERS: (1) We attach (different) fluorescent colours to each type of ddNTP (e.g., blue, red, ‘black’, green in this example). (2) We use gel electrophoresis to sort the fragments by size. The smallest fragments will represent DNA sequences terminating close to the primer. M11-3 9 ‘Fluorescent’ dideoxy sequencing is usually automated. Gel electrophoresis uses denaturing polyacrylamide gel (contains urea) to separate single-stranded DNA fragments by size. This type of gel gives very fine resolution, ability to distinguish fragments that differ by 1 base in size. As ddNTP-terminated fragments migrate in the gel, they pass a laser beam, that excites the fluorescent dyes, and a camera that records the flash of coloured light that results. M11-3 10 Pierce (2012) Genetics: A conceptual approach 4/e, Freedman & Co., New York. DNA sequencing state of the art 1990s Sanger dideoxy sequencing accurate but SLOW Originally, 4/tubes/sequence Later, automated fluorescent sequencing much faster, but still only ~100 samples/day (~650b/seq)  ~65,000 bases/day/machine. M11-3 11 Sanger dideoxy sequencing pros and cons: PROS Very accurate Relatively long sequencing reads (up to nearly 1,000b; although ~650b more common) Easy to do; can be automated. Low cost (for small numbers of samples). Continues to be used for all these reasons. CONS Too slow for many applications, such as genome sequencing! Costly when scaled up to acquire lots of data. Requires purification and preparation of each individual DNA sequence that is being studied. These limitations led to invention of other methods, so called ‘nextgeneration’ methods. M11-3 12 The Human Genome Project cost ~$3 billion (U.S.)!! Sequencing the first human genome was incredibly expensive. Many reasons, but main reason is that Sanger dideoxy sequencing is too slow and costly. A human genome sequence now costs 100 kb M11-3 29 Sequencing methods compared METHOD Massively parallel? Sequencing by synthesis? Single molecule? Chain terminator? Accuracy Read length SANGER NO YES NO YES, nonreversible 99.99% 6501,000 ILLUMINA YES YES NO YES, reversible up to 99.9% 75-600 NANOPORE YES NO YES NO up to ~98>99% -> 100 kb M11-3 30 Sequencing methods compared METHOD Massively parallel? Sequencing by synthesis? Single molecule? Chain terminator? Accuracy Read length SANGER NO YES NO YES, nonreversible 99.99% 6501,000 ILLUMINA YES YES NO YES, reversible up to 99.9% 75-600 NANOPORE YES NO YES NO up to ~98>99% -> 100 kb M11-3 31 Sequencing methods compared METHOD Massively parallel? Sequencing by synthesis? Single molecule? Chain terminator? Accuracy Read length SANGER NO YES NO YES, nonreversible 99.99% 6501,000 ILLUMINA YES YES NO YES, reversible up to 99.9% 75-600 NANOPORE YES NO YES NO up to ~98>99% -> 100 kb M11-3 32 Sequencing methods compared METHOD Massively parallel? Sequencing by synthesis? Single molecule? Chain terminator? Accuracy Read length SANGER NO YES NO YES, nonreversible 99.99% 6501,000 ILLUMINA YES YES NO YES, reversible up to 99.9% 75-600 NANOPORE YES NO YES NO up to ~98>99% -> 100 kb M11-3 33 Sequencing methods compared METHOD Massively parallel? Sequencing by synthesis? Single molecule? Chain terminator? Accuracy Read length SANGER NO YES NO YES, nonreversible 99.99% 6501,000 ILLUMINA YES YES NO YES, reversible up to 99.9%* 75-600 NANOPORE YES NO YES NO up to ~98>99%* -> 100 kb * Disadvantage of lower accuracy can be compensated for by sequencing same DNA sequence many times M11-3 34 Sequencing methods compared METHOD Massively parallel? Sequencing by synthesis? Single molecule? Chain terminator? Accuracy Read length SANGER NO YES NO YES, nonreversible 99.99% 6501,000 ILLUMINA YES YES NO YES, reversible up to 99.9% 75-600 NANOPORE YES NO YES NO up to ~98>99% -> 100 kb M11-3 35 Module 11-3 Important concepts Sanger dideoxy chain terminator method of DNA sequencing: how it works. ‘single sample’ vs. ‘massively parallel’ approaches to DNA sequencing. Illumina DNA sequencing: how it works. Nanopore DNA sequencing: basic principles. Similarities and differences between Sanger, Illumina and Nanopore. M11-3 36