Reagent for nucleic acid typing by primer extension

Details Reagent for nucleic acid typing by primer extension Reagent for nucleic acid typing by primer extension

1999-02-26

2003-03-25

Myers, Carla J. (Department: 1634)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S005000, C435S091200, C536S023100, C536S024330, C536S026100, C536S026120

Reexamination Certificate

active

06537748

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention relates to the field of nucleic acid sequence detection. The detection of nucleic acid sequences can be used in two general contexts. First, the detection of nucleic acid sequences can be used to determine the presence or absence of a particular genetic element. Second, the detection of nucleic acid sequences can be used to determine the specific type of a particular genetic element that is present. Variant genetic elements usually exist. Many techniques have been developed (1) to determine the presence of specific nucleic acid sequences, and (2) to compare homologous segments of nucleic acid sequence to determine if the segments are identical or if they differ at one or more nucleotides. Practical applications of these techniques include genetic disease diagnoses, infectious disease diagnoses, forensic techniques, paternity determinations, and genome mapping.
In general, the detection of nucleic acids in a sample and the subtypes thereof depends on the technique of specific nucleic acid hybridization in which the oligonucleotide probe is annealed under conditions of high stringency to nucleic acids in the sample, and the successfully annealed probes are subsequently detected (see Spiegelman, S.,
Scientific American
, Vol. 210, p. 48 (1964)).
The most definitive method for comparing DNA segments is to determine the complete nucleotide sequence of each segment. Examples of how sequencing has been used to study mutations in human genes are included in the publications of Engelke, et al.,
Proc. Natl. Acad. Sci. U.S.A
., 85:544-548 (1988) and Wong, et al.,
Nature
, 330:384-386 (1987). At the present time, it is not practical to use extensive sequencing to compare more than just a few DNA segments because the effort required to determine, interpret, and compare sequence information is time-consuming.
A commonly used screen for DNA polymorphisms arising from DNA sequence variation consists of digesting DNA with restriction endonucleases and analyzing the resulting fragments by means of Southern blots, as described by Botstein, et al.,
Am. J. Hum. Genet
., 32:314-331 (1980) and White, et al.,
Sci. Am
., 258:40-48 (1988). Mutations that affect the recognition sequence of the endonuclease will preclude enzymatic cleavage at that site, thereby altering the cleavage pattern of that DNA. DNAs are compared by looking for differences in restriction fragment lengths. A major problem with this method (known as restriction fragment length polymorphism mapping or RFLP mapping) is its inability to detect mutations that do not affect cleavage with a restriction endonuclease. Thus, many mutations are missed with this method. One study, by Jeffreys,
Cell
, 18:1-18 (1979), was able to detect only 0.7% of the mutational variants estimated to be present in a 40,000 base pair region of human DNA. Another problem is that the methods used to detect restriction fragment length polymorphisms are very labor intensive, in particular, the techniques involved with Southern blot analysis.
A technique for detecting specific mutations in any segment of DNA is described in Wallace, et al.,
Nucl. Acids Res
., 9:879-894 (1981). It involves hybridizing the DNA to be analyzed (target DNA) with a complementary, labeled oligonucleotide probe. Due to the thermal instability of DNA duplexes containing even a single base pair mismatch, differential melting temperature can be used to distinguish target DNAs that are perfectly complementary to the probe from target DNAs that differ by as little as a single nucleotide. In a related technique, described in Landegren, et al.,
Science
, 41:1077-1080 (1988), oligonucleotide probes are constructed in pairs such that their junction corresponds to the site on the DNA being analyzed for mutation. These oligonucleotides are then hybridized to the DNA being analyzed. Base pair mismatch between either oligonucleotide and the target DNA at the junction location prevents the efficient joining of the two oligonucleotide probes by DNA ligase.
A. Nucleic Acid Hybridization
The base pairing of nucleic acids in a hybridization reaction forms the basis of most nucleic acid analytical and diagnostic techniques. In practice, tests based only on parameters of nucleic acid hybridization function poorly in cases where the sequence complexity of the test sample is high. This is partly due to the small thermodynamic differences in hybrid stability, generated by single nucleotide changes, and the fact that increasing specificity by lengthening the probe has the effect of further diminishing this differential stability. Nucleic acid hybridization is, therefore, generally combined with some other selection or enrichment procedure for analytical and diagnostic purposes.
Combining hybridization with size fractionation of hybridized molecules as a selection technique has been one general diagnostic approach. Size selection can be carried out prior to hybridization. The best known prior size selection technique is Southern Blotting (see Southern, E.,
Methods in Enzymology
, 69:152 (1980). In this technique, a DNA sample is subjected to digestion with restriction enzymes which introduce double stranded breaks in the phosphodiester backbone at or near the site of a short sequence of nucleotides which is characteristic for each enzyme. The resulting heterogeneous mixture of DNA fragments is then separated by gel electrophoresis, denatured, and transferred to a solid phase where it is subjected to hybridization analysis in situ using a labeled nucleic acid probe. Fragments which contain sequences complementary to the labeled probe are revealed visually or densitometrically as bands of hybridized label. A variation of this method is Northern Blotting for RNA molecules. Size selection has also been used after hybridization in a number of techniques, in particular by hybrid protection techniques, by subjecting probe
ucleic acid hybrids to enzymatic digestion before size analysis.
B. Polymerase Extension of Duplex Primer:template Complexes
Hybrids between primers and DNA targets can be analyzed by polymerase extension of the hybrids. A modification of this methodology is the polymerase chain reaction in which the purification is produced by sequential hybridization reactions of anti-parallel primers, followed by enzymatic amplification with DNA polymerase (see Saiki, et al.,
Science
239:487-491 (1988)). By selecting for two hybridization reactions, this methodology provides the specificity lacking in techniques that depend only upon a single hybridization reaction.
It has long been known that primer-dependent DNA polymerases have, in general, a low error rate for the addition of nucleotides complementary to a template. This feature is essential in biology for the prevention of genetic mistakes which would have detrimental effects on progeny. The specificity inherent in this enzymological reaction has been widely exploited as the basis of the “Sanger” or dideoxy chain termination sequencing methodology which is the ultimate nucleic acid typing experiment. One type of Sanger DNA sequencing method makes use of mixtures of the four deoxynucleoside triphosphates, which are normal DNA precursors, and one of the four possible dideoxynucleoside triphosphates, which have a hydrogen atom instead of a hydroxyl group attached to the 3′ carbon atom of the ribose sugar component of the nucleotide. DNA chain elongation in the 5′ to 3′ direction (“downstream”) requires this hydroxyl group. As such, when a dideoxynucleotide is incorporated into the growing DNA chain, no further elongation can occur. With one dideoxynucleotide in the mixture, DNA polymerases can, from a primer:template combination, produce a population of molecules of varying length, all of which terminate after the addition of one out of the four possible nucleotides. The series of four independent reactions, each with a different dideoxynucleotide, generates a nested set of fragments, all starting at the same 5′ terminus of the priming DNA molecule and terminating at all possible 3&prim

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