Methods and Compositions for Efficient Nucleic Acid Sequencing

Abstract

Disclosed are novel methods and compositions for rapid and highly efficient nucleic acid sequencing based upon hybridization with two sets of small oligonucleotide probes of known sequences. Extremely large nucleic acid molecules, including chromosomes and non-amplified RNA, may be sequenced without prior cloning or subcloning steps. The methods of the invention also solve various current problems associated with sequencing technology such as, for example, high noise to signal ratios and difficult discrimination, attaching many nucleic acid fragments to a surface, preparing many, longer or more complex probes and labelling more species.

Description

[0001] The present application is a continuation-in-part of co-pending U.S. patent application Ser. No. 08 / 303,058, filed Sep. 8, 1994; which is a continuation-in-part of U.S. patent application Ser. No. 08 / 127,420, filed Sep. 27, 1993; the entire text and figures of which disclosures are specifically incorporated herein by reference without disclaimer.[0002] The U.S. Government owns rights in the present invention pursuant to Department of Energy grant LDRD 03235 and Contract No. W-31-109-ENG-38 between the U.S. Department of Energy and The University of Chicago, representing Argonne National Laboratory.BACKGROUND OF THE INVENTION [0003] 1. Field of the Invention [0004] The present invention generally relates to the field of molecular biology. The invention particularly provides novel methods and compositions to enable highly efficient sequencing of nucleic acid molecules. The methods of the invention are suitable for sequencing long nucleic acid molecules, including chromosomes an...

AI Technical Summary

Benefits of technology

The present invention provides new methods and compositions for sequencing nucleic acids by using small oligonucleotide probes of known sequence. These methods allow for high discriminatory sequencing of very large nucleic acid molecules without prior cloning, subcloning, or amplification. The methods can be conducted using sequential hybridization or simultaneous hybridization techniques, and can be applied to both chromosomal material and RNA. The invention also includes methods for detecting and measuring the sequences of nucleic acid fragments using labeled probes and automated synthesis. The new methods and compositions offer improved accuracy and efficiency in sequencing nucleic acids and have applications in areas such as molecular biology and genomic analysis.

Problems solved by technology

However, progress in this area is generally both slow and costly.

Both methods are time-consuming and laborious.

However, although SBH has the potential for increasing the speed with which nucleic acids can be sequenced, all current SBH methods still suffer from several drawbacks.

Unfortunately, both of these SBH formats have several limitations, particularly the requirement for prior DNA cloning steps.

In Format 1, other significant problems include attaching the various nucleic acid pieces to be sequenced to the solid surface support or preparing a large set of longer probes.

In Format 2, major problems include labelling the nucleic acids of unknown sequence, high noise to signal ratios that generally result, and the fact that only short sequences can be determined.

Further problems of Format 2 include the secondary structure formation that prevents access to some targets and the different conditions that are necessary for probes with different GC contents.

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