Primer extension methods for detecting nucleic acids

Details Primer extension methods for detecting nucleic acids Primer extension methods for detecting nucleic acids

1998-04-27

2003-05-20

Whisenant, Ethan C. (Department: 1634)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S006120, C435S091100, C436S094000, C536S023100, C536S024300, C536S024330

Reexamination Certificate

active

06566101

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to oligonucleotide primer extension methods for identifying a single nucleotide in a nucleic acid sample. Methods of the invention are useful for disease diagnosis by detecting and identifying the presence of genetic mutations or disease-causing microorganisms in biological samples.
BACKGROUND OF THE INVENTION
The knowledge of molecular defects causative of diseases, such as inherited disorders and cancer, is increasing rapidly. Inherited diseases thought to be caused by genetic mutations include sickle cell anemia, &agr;- and &bgr;-thalassemias, phenylketonuria, hemophilia, &agr;
i
-anti-trypsin deficiency, and cystic fibrosis. Sickle cell anemia, for example, is reported to result from homozygosity resulting from a single base pair substitution in the sixth codon of the &bgr;-globin gene. Antonarakis,
New England J. Med
., 320: 153-163 (1989). Mutations in the insulin receptor gene and in the insulin-responsive glucose transporter gene have been detected in insulin-resistant diabetes. Krook et al.,
Human Molecular Genetics
, 1: 391-396 (1992).
Cancer has been associated with genetic mutations in a number of oncogenes and tumor suppressor genes. Duffy,
Clin. Chem
., 41: 1410-1413 (1993). For example, point mutations in the ras genes have been shown to convert those genes into transforming oncogenes. Bos et al.,
Nature
, 315: 726-730. Mutations and the loss of heterozygosity at the p53 tumor suppressor locus have been correlated with various types of cancer. Ridanpaa et al.,
Path. Res. Pract
., 191: 399-402 (1995); Hollstein et al.,
Science
, 253: 49-53 (1991). In addition, the loss or other mutation of the apc and dcc tumor suppressor genes has also been associated with tumor development. Blum,
Europ. J. Cancer
, 31A: 1369-1372 (1995). Those mutations can serve as markers for early stages of disease and for predisposition thereto. Early diagnosis is not only important for successful treatment, but can also lead to prevention or treatment before chronic symptoms occur.
Colorectal cancer is an example of a disease that is highly curable if detected early. With early detection, colon cancer may be effectively treated by, for example, surgical removal of the cancerous tissue. Surgical removal of early-stage colon cancer is usually successful because colon cancer begins in cells of the colonic epithelium and is isolated from the general circulation during its early stages. Thus, detection of early mutations in colorectal cells would greatly increase survival rate. Current methods for detection of colorectal cancer focus on extracellular indicia of the presence of cancer, such as the presence of fecal occult blood or carcinoembryonic antigen circulating in serum. Such extracellular indicia typically occurs only after the cancer has become invasive. At that point, colorectal cancer is very difficult to treat.
Methods have been devised to detect the presence of mutations within disease-associated genes. One such method is to compare the complete nucleotide sequence of a sample genomic region with the corresponding wild-type region. See, e.g., Engelke et al.,
Proc. Natl. Acad. Sci, U.S.A
., 85: 544-548 (1988); Wong et al.,
Nature
, 330: 384-386 (1988). However, such methods are costly, time consuming, and require the analysis of multiple clones of the targeted gene for unambiguous detection of low-frequency mutations. As such, it is not practical to use extensive sequencing for large-scale screening of genetic mutations.
A variety of detection methods have been developed which exploit sequence variation in DNA using enzymatic and chemical cleavage techniques. A commonly-used screen for DNA polymorphisms consists of digesting DNA with restriction endonucleases and analyzing the resulting fragments by means of southern blots, as reported 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 the DNA. Sequences are compared by looking for differences in restriction fragment lengths. A 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. One study reported that only 0.7% of the mutational variants estimated to be present in a 40,000 base pair region of human DNA were detected using RFLP analysis. Jeffreys,
Cell
, 18: 1-18 (1979).
Single base mutations have been detected by differential hybridization techniques using allele-specific oligonucleotide (ASO) probes. Saiki et al.,
Proc. Natl. Acad. Sci. USA
, 86: 6230-6234 (1989). Mutations are identified on the basis of the higher thermal stability of the perfectly-matched probes as compared to mismatched probes. Disadvantages of this approach for mutation analysis include: (1) the requirement for optimization of hybridization for each probe, and (2) the nature of the mismatch and the local sequence impose limitations on the degree of discrimination of the probes. In practice, tests based only on parameters of nucleic acid hybridization function poorly when the sequence complexity of the test sample is high (e.g., in a heterogeneous biological sample). This is partly due to the small thermodynamic differences in hybrid stability generated by single nucleotide changes. Therefore, nucleic acid hybridization is generally combined with some other selection or enrichment procedure for analytical and diagnostic purposes.
In enzyme-mediated ligation methods, a mutation is interrogated by two oligonucleotides capable of annealing immediately adjacent to each other on a target DNA or RNA molecule, one of the oligonucleotides having its 3′ end complementary to the point mutation. Adjacent oligonucleotide sequences are only covalently attached when both oligonucleotides are correctly base-paired. Thus, the presence of a point mutation is indicated by the ligation of the two adjacent oligonucleotides. Grossman et al.,
Nucleic Acid Research
, 22: 4527-4534 (1994). However, the usefulness of this method for detection is compromised by high backgrounds which arise from tolerance of certain nucleotide mismatches or from non-template directed ligation reactions. Barringer et al.,
Gene
, 89: 117-122 (1990).
A number of detection methods have been developed which are based on a template-dependent, primer extension reaction. These methods fall essentially into two categories: (1) methods using primers which span the region to be interrogated for the mutation, and (2) methods using primers which hybridizes proximally and upstream of the region to be interrogated for the mutation.
In the first category, Caskey and Gibbs [U.S. Pat. No. 5,578,458] report a method wherein single base mutations in target nucleic acids are detected by competitive oligonucleotide priming under hybridization conditions that favor the binding of the perfectly-matched primer as compared to one with a mismatch. Vary and Diamond [U.S. Pat. No. 4,851,331] described a similar method wherein the 3′ terminal nucleotide of the primer corresponds to the variant nucleotide of interest. Since mismatching of the primer and the template at the 3′ terminal nucleotide of the primer inhibits elongation, significant differences in the amount of incorporation of a tracer nucleotide result under normal primer extension conditions.
It has long been known that primer-dependent DNA polymerases have, in general, a low replication error rate. This feature is essential for the prevention of genetic mistakes which would have detrimental effects on progeny. Methods in a second category exploit the high fidelity inherent in this enzymological reaction. Detection of mutations is based on primer extension and incorporation of detectable, chain-terminating nucleoside triphosphates. The high fidelity of DNA polymerases ensures specific incorporation of the co

Affiliated with

Also associated with

Rating

Primer extension methods for detecting nucleic acids does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Primer extension methods for detecting nucleic acids, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Primer extension methods for detecting nucleic acids will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3081438