Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-06-13
2003-10-28
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06638722
ABSTRACT:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not applicable.
REFERENCE TO A “MICROFICHE APPENDIX”
Not applicable.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present disclosure relates generally to the field of DNA amplification and more particularly to the field of amplifying any stretch of DNA in a sequence-independent manner.
2. Description of Related Art
The following description includes information that may be useful in understanding the present disclosure. It is not an admission that any of the information provided herein is prior art, or relevant, to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.
It is well known that there is often an association between genetic variation and phenotype manifestation. Genetic variations and their associated phenotypes are studied using various methods of genotyping genomic DNA. A Single Nucleotide Polymorphism (SNP) is a single nucleotide variation at a specific location in the genome of different individuals. SNPs are stable genetic variations frequently found in genes, and contribute to the wide range of phenotypic variations found in organisms. SNP genotyping is useful in developing detailed genetic and physical maps of chromosomes. Genotyping densely distributed SNP markers across the different chromosomes of an individual can help reveal statistically significant correlations between chromosomal loci and phenotypic expression. Extensive genotyping, however, requires not only a simple and rapid way for obtaining, shipping, storing, and sorting large amounts of genetic material, but also convenient and high-throughput methods for extracting large quantities of DNA from these samples.
There are a variety of available methods for obtaining and storing tissue and/or blood samples. These alternatives allow tissue and blood samples to be stored and transported in a form suitable for the recovery of genomic DNA from the samples for genotype analysis. DNA samples can be collected and stored on a variety of solid mediums, including Whatmann® paper, Guthrie cards, tubes, swabs, filter paper, slides, or other containers. When whole blood is collected on filter paper, for example, it can be dried and stored at room temperature.
One known and more frequently used method for securing and storing DNA is described in U.S. Pat. No. 5,496,562. This method involves storing dried animal blood samples on chemically treated filter paper, called FTA paper, that protects genomic DNA from degrading (commercially available as FTA™ paper by Whatman®). FTA paper is light weight and easy to store, which makes it a popular choice for collecting genetic material and samples. Samples on FTA paper are conveniently stored and shipped at room temperature.
All of the materials available to those of skill in the art for storing blood or other tissues containing DNA have limitations. For example, the amount of tissue or blood collected may be very limited, which makes wide-scale and high-throughput genotyping impractical and expensive. For example, despite the widespread use of FTA paper, its usefulness is limited because the stored bloodstains contain only a small amount of genomic DNA. A 6.0 cm
2
piece of FTA paper only preserves approximately 100 &mgr;l of blood, equivalent to approximately 1.0 &mgr;g of DNA. While it is possible to extract genomic DNA from a larger piece of FTA paper, the size of the paper makes it cumbersome to manipulate in the small wells of a 96-well plate or a 384-well plate, both of which are important tools for high-throughput screening of large numbers of DNA samples. Therefore, the usefulness of a tool like FTA paper has been restricted to low-volume genotyping.
The limited amount of DNA stored on FTA paper also makes it impractical for genotyping multiple polymorphisms and genetic loci in a single organism. The FTA paper sample can be cut into smaller pieces for genotyping multiple SNPs; a small circle of 1.0-2.0 mm
2
diameter of the sample contains about 1-5 ng of genomic DNA, which is sufficient for one polymerase chain reaction (PCR). But this approach is undesirable because it requires repetitive cutting, sorting, and extracting of the FTA paper, which is not only tedious but also prone to human error. For a genomic scan of hundreds or thousands of SNPs, the task of cutting and analyzing DNA samples stored on FTA paper is an insurmountable barrier for researchers.
Additionally, the strong adherence between DNA and FTA paper makes DNA extraction for analysis difficult. Although proteinase K and endonuclease digestion can facilitate DNA release as suggested by the manufacturer, this approach is too complicated and expensive for high-throughput operations. The commercially available FTA Purification Reagent, which can be used to prepare DNA stored on FTA paper for analysis by PCR™ yields inconsistent results. For example, often no specific DNA amplification is achieved after the DNA sample is processed using this reagent, which is unacceptable in a high-throughput operation. The manufacturer also suggests that the strong adhesion of DNA to FTA paper allows for repeated genotyping of DNA stored on FTA paper. Notwithstanding the fact that PCR efficacy for “recycled” FTA paper has not been fully tested, cleaning the tiny FTA papers between consecutive SNP PCRs is impractical for high-throughput processing. The small pieces of floating filter paper are difficult to wash by conventional aspiration, and they tend to clog aspiration needles or pipette tips. Further, small pieces are easily lost during the cleaning process. Finally, repeated pipetting of PCR products has an associated risk of cross contamination among different wells.
The shortcomings associated with small samples of blood or tissue from an organism are overcome by efficient methods of whole genomic DNA amplification. For example, whole genomic DNA amplified from the small amounts of DNA sample stored on FTA paper could be used in multiple PCR reactions to extensively genotype various polymorphisms such as SNPs found in a single organism in a high-throughput screening process. Nevertheless, while several methods for whole genome amplification have been proposed and successfully used for various applications in the past, these methods are generally inefficient, complex, and expensive. Therefore, the need exists for a simple and cost effective way of amplifying genomic DNA from small samples of blood or tissue.
One of the first methods for amplifying DNA was the linker adaptor-mediated PCR (LAM-PCR) approach, which has been applied to microdissected chromosomes (Zhou et al.,
Bio Techniques
28:766-774, 2000; Albani et al.,
Plant J
4(5):899-903, November 1993), yeast artificial chromosome (YAC) DNA (Sutcliffe et al.,
Genomics
13(4):1303-6, 1992), and genomic DNA (Kinzler et al.,
Nucleic Acids Res
25:17(10):3645-53, May 1989). In this approach, the starting DNA is first digested with a restriction enzyme, usually an enzyme with a four base recognition sequence. After inactivation of the restriction enzyme, a known sequence (either an adaptor or a synthetic linker) is ligated to the ends of the DNA fragments generated by the restriction-enzyme digest, providing primer binding sites for PCR amplification. The DNA can then be amplified by PCR using primers that are complementary to the sequence of the adaptor or linker.
Unfortunately, the usefulness of LAM-PCR is limited because it involves multiple steps, including DNA fragmentation, adaptor or linker ligation, and PCR amplification. These steps make this process both laborious and expensive for high-throughput genotyping. An additional shortcoming of this method is that sequences that do not contain the recognition sequence of the restriction enzyme used at appropriately spaced intervals will not be amplified by PCR because the regions will be too long to amplify. This method is also time-consuming and cumbersome because of the extensive manipulations of DNA necessary to attach the known sequences to both ends of the fragments, e
Davis Scott
Gregg Keqin
Ji Wan
Kemppainen Jon
Reus Bonnie
Horlick Kenneth R.
Invitrogen Corporation
Vinson & Elkins L.L.P.
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