Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1994-12-09
2000-07-11
Degen, Nancy
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 41, 4351721, 536 2433, 536 253, C12Q 168, C12P 1934, C07N 2100
Patent
active
060870958
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method for sequencing DNA. In particular, the present invention concerns a method for the automated sequencing of large fragments of DNA.
DNA sequence analysis has become one of the most important tools available to the molecular biologist. Current sequencing technology allows sequence data to be obtained from virtually any DNA fragment. This has allowed not only the sequencing of entire genes and other genomic sequences but also the identification of the sequence of RNA transcripts, by the sequencing of cDNA. Currently, emphasis is being placed on genomic sequencing in order to determine the DNA sequence of entire genomes. Ultimately, it is hoped that the sequence of the human genome will be deciphered.
Traditional DNA sequencing techniques share three essential steps in their approaches to sequence determination. Firstly, a multiplicity of DNA fragments are generated from a DNA species which it is intended to sequence. These fragments are incomplete copies of the DNA species to be sequenced. The aim is to produce a ladder of DNA fragments, each a single base longer than the previous one. This can be achieved by selective chemical degradation of multiple copies of the DNA species to be sequenced, as in the Maxam and Gilbert method (A. Maxam and W. Gilbert, PNAS 74, p. 560, 1977). Alternatively, the DNA species can be used as a template for a DNA polymerase to produce a number of incomplete clones, as in the Sanger method (F. Sanger, S. Nicklen and A. Coulson, PNAS 74, p. 5463, 1977). These fragments, which differ in respective length by a single base, are then separated on an apparatus which is capable of resolving single-base differences in size. A thin polyacrylamide gel is invariably used in this process. The third and final step is the determination of the nature of the base at the end of each fragment. When ordered by the size of the fragments which they terminate, these bases represent the sequence of the original DNA species.
Determination of the nature of each base is achievers by previously selecting the terminal base of each fragment. In the Sanger method, for example, dideoxy nucleoside triphosphates (ddNTPs) are used to selectively terminate growing DNA clones at an A, C, G or T residue. This means that four separate reactions need to be performed for each sequencing exercise, each in a separate tube using a different ddNTP. In one tube, therefore, each labelled fragment will terminate with an A residue, while in the next tube with a C residue, and so on. Separation of each croup of fragments side-by-side on a polyacrylamide gel will show the sequence of the template by way of the relative size of the individual fragments.
In the Maxam and Gilbert method, on the other hand, the selectivity is achieved during the chemical degradation process. Chemicals are used which cleave DNA strands at A only, C only, G and A or T and C. Use of limiting concentrations of such chemicals allows partial digestion of the DNA species. As in the Sanger method, four separate reactions must be performed and the products separated side-by-side on a polyacrylamide gel.
The disadvantages of these prior art methods are numerous. They require a number of complex manipulations to be performed, in at least four tubes. They are susceptible to errors due to the formation of secondary structures in DNA, or other phenomena that prevent faithful replication of a DNA template in the Sanger method or which cause base-specificity to be lost by the chemical reactants of the Maxam and Gilbert method. The most serious problems, however, are caused by the requirement for the DNA fragments to be size-separated on a polyacrylamide gel. This process is time-consuming, uses large quantities of expensive chemicals, and severely limits the number of bases which can be sequenced in any single experiment, due to the limited resolution of the gel. Furthermore, reading the gels in order to extract the data is labour-intensive and slow.
A number of improvements have been effected to these sequencing methods in
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Drmanac et al., "Sequencing of Megabase Plus DNA by Hybridization: . . . ", Genomics 4:114-128, 1989.
Salmeron et al., "Imaging of Biomolecules with the Scanning Tunneling Microscope: . . . ", J. Vac. Sci. Tech. 8:635, Jan./Feb. 1990.
Maxam et al., "A new method for sequencing DNA", Proc. Natl. Sci. USA 74:560-564, Feb. 1977.
Drmanac et al., "Reliable Hybridization of Oligonucleotides as Short as Six Nucleotides", DNA and Cell Biology 9:527, Nov. 1990.
Sanger et al., "DNA sequencing with chain-terminating inhibitors", Proc. Natl. Acad. Sci. USA 74:5463-5467, Dec. 1977.
Bains et al., "A Novel Method for Nucleic Acid Sequence Determination", J. Theor. Biol. 135:303-307, 1988.
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P.A. Pevzner, "1-Tuple DNA Sequencing:Computer Analysis", J. Biom. Str. & Dyn. 7:63, 1989.
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Brenner Sydney
Rosenthal Andre
Degen Nancy
McGarry Sean
Medical Research Council
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