Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-11-04
2002-04-30
Sisson, Bradley L. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S006120, C436S094000, C436S172000, C436S800000, C436S805000, C536S023100, C536S024300, C536S025300
Reexamination Certificate
active
06379889
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to multiplexed chromatographic methods. Such methods can be used for detecting mutations within a population of nucleic acid samples. They can also be used for genotyping and haplotyping.
BACKGROUND OF THE INVENTION
Deciphering the genetic code and the establishment of the structure of deoxyribonucleic acid (DNA) in the early 1960s initiated a revolution in modem biology. Since that time, numerous methods have been developed for the isolation, analysis, and manipulation of nucleic acid samples.
One such method developed for the analysis of nucleic acid samples is polymerase chain reaction amplification (referred to herein as “PCR”). PCR is an in vitro method for replicating a defined (or target) DNA molecule to increase the amount of total DNA for subsequent analysis, such as sequencing, Northern and Southern hybridizations, and the like. Typically, the amount of total DNA increases exponentially, i.e., it is amplified. Thus, PCR can be utilized in connection with a variety of techniques when it is desirable to manipulate and analyze genetic information of a DNA molecule that may be in low copy numbers. For example, PCR may be used in connection with cloning genes, sequencing, genome mapping, site directed mutagenesis, diagnostic assays, environmental monitoring, to name a few.
Due to the vast amount of genetic information that is capable of being generated and gathered, intense efforts are underway to develop new and faster methods of DNA detection, sizing, quantification, sequencing, and gene identification including the mapping of human disease genes. Although the efficiency of these processes has been improved by automation, more efficient and less expensive methods must still be developed to efficiently carry out genomic-scale DNA analyses.
The detection of polymorphisms is becoming increasingly important, particularly in gene mapping. Although the majority of DNA in higher organisms is identical in sequence among the chromosomes of different individuals, a small fraction of DNA is variable or polymorphic in sequence. It is this variation which is the essence of genetic science and human diversity. Mutations arise either due to environmental effects or randomly during replication as a change in the sequence of a gene, with different mutations having differing consequences. In fact, single base pair changes, called single nucleotide polymorphisms (SNPs) are frequent in the human genome. The level of genetic variation between two individual sequences is estimated to be on average one difference per 1,000 base pairs. Based on this estimate, the average amount of genomic variation between individuals is about 3 million base pairs. It is this normal polymorphism, which provides the basis for some of the emerging gene localization strategies.
As the sequences of greater numbers of genes are identified, the detection of specific polymorphisms in such genes and the correlation to specific diseases can provide an invaluable tool in the screening and detection of diseases. Diagnostic screening methods for polymorphisms are also useful in the detection of inherited diseases in which either a single point mutation or a few known mutations account for all cases (e.g., sickle cell disease). Presently, over 200 genetic disorders can be diagnosed using recombinant DNA techniques. Such techniques have also been used for other purposes, such as for forensic screening.
Presently used methods for screening for polymorphic sites within a gene include single-stranded conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), RNase A cleavage, chemical cleavage, allele specific oligonucleotides (ASOs), ligase mediated detection of mutations, and denaturing high performance liquid chromatography.
Briefly, in single-stranded conformation polymorphism (SSCP), DNA is denatured and then immediately run on a non-denaturing gel. The secondary structures of wild-type strands or mutant single strands differing by a single base are usually sufficiently different to result in different migration rates on polyacrylamide gels.
In denaturing gradient gel electrophoresis (DGGE), either homoduplex or heteroduplex double stranded DNA is electrophoresed under denaturing conditions of increasing concentration until the last domain is denatured, and migration of the DNA through the gel is retarded. DNA sequences differing by a single base pair migrate at different rates along the gel, thereby allowing detection of a polymorphic site, if present.
RNase A cleavage utilizes the enzyme ribonuclease A to cut RNA-DNA hybrids wherever there is a mismatch between a nucleotide in the RNA strand and the corresponding nucleotide in the DNA strand. The chemical cleavage method is based upon a similar principle but uses hydroxylamine and osmium tetroxide to distinguish between mismatched C or T nucleotides, respectively. The position of the mismatch (e.g., the mutation) is defined by sizing on gel electrophoresis after cleavage at the reactive position by piperidine.
Allele-specific oligonucleotide probes (ASOs) are probes that are designed to hybridize selectively to either a normal or a mutant allele, where the probes are developed to distinguish between the normal and mutant sequence. This is done by altering the stringency of hybridization to a level at which each of the oligonucleotides will anneal stably only to the sequence to which it is perfectly complementary and not to the sequence with which it has the single mismatch.
The ligase-mediated method for detecting mutations makes use of the fact that the ends of two single strands of DNA must be exactly aligned for DNA ligase to join them. In utilizing this technique, oligonucleotides complementary to the target sequence, 5′ to and including the mutation site, are synthesized and labeled. A third oligonucleotide complementary to the common sequence 3′ to the mutation site is synthesized and also labeled. The oligonucleotides are then hybridized to strands of the target. In cases in which the 5′ and 3′ oligonucleotides form a flush junction that can be joined by DNA ligase, ligation occurs. However, a single base pair mismatch occurring between the normal 5′ oligonucleotide and the mutation site is sufficient to prevent the ligase from acting and can readily be detected.
A common approach to analysis of DNA polymorphisms relies on variations in the lengths of DNA fragments produced by restriction enzyme digestion. The polymorphisms identified using this approach are typically referred to as restriction fragment length polymorphisms or RFLPs. Polymorphisms involving variable numbers of tandemly repeated DNA sequences between restriction enzyme sites, typically referred to as microsatellites or variable numbers of tandem repeats (VNTRs), have also been identified.
While existing methods may locate polymorphic sites, point mutations, insertions and deletions on a gene, many of these methods are generally time consuming, necessitate multiple handling steps, require product purification, are not readily adaptable to automation, have limitations in sensitivity and accuracy, and are typically limited to detection in small-sized nucleic acid fragments.
Furthermore, existing methods typically do not yield haplotype information (i.e., linked polymorphism) without the use of multiple, and often complicated, steps that may incorporate toxic chemicals. See, for example, Verpy et al.,
Proc. Natl. Acad. Sci., USA,
95, 1873-1877 (1994).
Denaturing high performance liquid chromatography for separating heteroduplex (double-stranded nucleic acid molecules having less than 100% sequence complementarity) and homoduplex (double-stranded nucleic acid molecules having 100% sequence complementarity) nucleic acid samples (e.g., DNA or RNA) in a mixture is described in U.S. Pat. No. 5,795,976 (Oefler et al.). In the separation method, a mixture containing both heteroduplex and homoduplex nucleic acid samples is applied to a stationary reversed phase support. The sample mixture is then elute
Apffel, Jr. James A.
Hahnenberger Karen M.
Kronick Mel N.
Verhoef Martin
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