Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1998-05-15
1999-10-26
Horlick, Kenneth R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
436 94, C12Q 168, G01N 3348
Patent
active
059726121
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to an improved method of nucleic acid sequencing based on the detection and measurement of hybridization interactions.
BACKGROUND OF THE INVENTION
The generation of DNA sequence information has dramatically increased due to the large programs for sequencing of the human genome. Today that work is mainly done by traditional gel electrophoretic separation of DNA fragments terminated at different positions, either enzymatically (dideoxy chain termination method according to Sanger et al., Proc. Natl. Acad. Sci. USA 74: 5463-5467 (1977)) or chemically (chemical degradation method according to Maxam and Gilbert, Proc. Natl. Acad. Sci. USA 74: 560-564). These systems are, however, both time- and labour-intensive.
There is therefore a general need for more effective methods for de novo sequencing of DNA as well as for repeated sequencing of known sequences for analysis of mutations, such as point mutations. The mutation analysis will increase as more information will be gathered about the correlation between different diseases and mutations and also due to the need to verify deliberately introduced mutations in biotechnology work.
Sequencing by hybridization (SBH) (see e.g. Drmanac et al., Genomics 4: 114; Strazoski et al., Proc. Natl. Acad. Sci. USA 88: 10089 (1991); Bains and Smith, J. Theoretical Biol. 135: 303 (1988); and U.S. Pat. No. 5,202,231) has become an interesting alternative to traditional sequencing with a potential for higher through-put of information. This type of system utilizes the information obtained from multiple hybridizations of the polynucleotide of interest, using short oligonucleotides to determine the nucleic acid sequence. However, there are several technical problems associated with this technology. For example, while today there are ways to build arrays of oligonucleotides on a chip based on the synthesis of oligoprobes and photolitographic techniques, it is still complicated to provide on a chip the large set of oligonucleotide probes required for determining a random nucleic acid sequence. Further, the detection of interaction of labelled target DNA is normally done by fluorescent or radioactivity measurements. Such detection is dependent on washing of the chip to get rid of residual labelled target molecules and the oligoprobes must therefore bind rather strongly to the target molecules. There are also problems with the binding of oligoprobes with a single base mismatch in combination with the different sensitivity to washing conditions dependent on base pair composition, G:C being more stable than A:T. One attempt to overcome such problems is to use tetraalkylammonium salts that eliminate the difference in stability of G:C and A:T base pairs.
Even if differences in base composition can be compensated for, the whole SBH procedure is based on interaction, washing, and detection of hybridized target DNA and oligoprobe. The conditions for the hybridization thus have to be adjusted for a stable hybridization which can be detected only after several washing cycles. Dependent on the position of the mismatch of single bases, base composition, oligoprobe length and temperature, there will be several hybridizations of oligomers that will show up as weaker binding and such interactions will be problematic to determine. Temperature and salt gradients elution have been suggested but are difficult to elaborate technically.
Due to the conditions needed for hybridization there is also always a potential risk for the target DNA to hybridize to itself due to complementary regions of the DNA.
A major disadvantage of SBH is, however, that the information is exclusively based on short-range information and the fact that overlaps are unique. Success is dependent on whether or not there are repeated sequences in the nucleic acid to be analysed. The need and importance of repeated sequences are known from several situations, not least in the analysis of genes like, for example, the gene for Huntington's disease where repeated sequences and the am
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Stimpson et al., Proc. Natl. Acad. Sci. USA 92, 6379-6383 (Jul. 1995).
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Stimpson et al., Genetic Analysis Biomolec. Eng. 13, 73-80 (1996).
Gotoh et al., DNA Res. 2(6), 285-293 (Dec. 1995)(abstract only).
Malmqvist Magnus
Persson Bjorn
Biacore AB
Horlick Kenneth R.
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