Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-10-14
2001-02-13
Marschel, Ardin H. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S025300, C536S025400
Reexamination Certificate
active
06187539
ABSTRACT:
FIELD OF THE INVENTION
The present invention concerns an improved method for the analysis and detection of nicks in double stranded DNA.
BACKGROUND OF THE INVENTION
The term “nick” is defined herein to mean a double stranded (ds) DNA fragment wherein one of the strands is contiguous and the complimentary strand contains at least one break, wherein two adjacent bases are not covalently linked. “Nicks” in DNA fragments occur for a variety of reasons. For example, when long DNA fragments are constructed by enzymatic ligation of shorter strands, some fragments may not be completely ligated. Important and widely used products of DNA ligation comprise commercially produced DNA sequencing “ladders” (BioRad, Inc., Richmond, Calif.; Life Technologies, Inc., Germantown, Md.). DNA “ladders” are mixtures of DNA fragments, wherein the fragments comprise a defined range of base pair lengths and the fragments in the ladder differ by a constant base pair increment. For example, a 100 base pair (100 bp) ladder contains DNA fragments which differ by 100 bp increments over a range of 100 bp to 3,000 bp, i.e., 1000 bp, 200 bp, 300 bp, 400 bp . . . 3,000 bp. Such DNA ladders are used as base pair length standards to calibrate electrophoresis gels. The accuracy of such ladders is of critical importance, since defective or impure ladders may lead to incorrect interpretation of sample results when compared to the standard base pair ladders. However, while a ladder containing nicks might be usable as a standard for gel electrophoresis, Applicants have found that such a ladder is not suitable for use as a standard using the more accurate and sensitive separation methods described herein.
Nicks are also formed when an enzyme which recognizes a base pair mismatch (mutation) in a heteroduplex, binds within the vicinity of a mutation and cleaves one strand of the DNA duplex which contains a non-complimentary base while leaving the other strand intact. Many such enzymes are known in the art and they are the basis of one form of mutation detection. For a comprehensive description of this subject see U.S. Pat. No. 5,763,178 to Chirikjian (1998). This reference and the references contained therein are incorporated in their entireties herein.
A nick in dsDNA cannot be detected by either gel or capillary electrophoresis of native DNA directly. Nicked strands can be detected, however, using denaturing polyacrylamide gels (
Molecular Cloning
, 2
nd
Ed. Sambrook et al. eds. Cold Spring Harbor Laboratory Press, 1989, incorporated herein by reference). Gel electrophoresis can also separate and detect nicked double stranded fragments which have been tagged with fluorescent or radioactive probes. However, this approach is costly and very labor intensive in that it requires the preparation of DNA fragments tagged with expensive probes. Methods which depend on gel electrophoresis (GEP) for separation of DNA fragments are subject to inherent deficiencies. This separation method is difficult to implement, not always reproducible, not accurate, difficult to quantify, and routinely takes five hours or more to complete (not counting set up time).
Other limitations in using GEP are related to the development and interpretation of bands on gels. The bands are often curved rather than straight, their mobility and shape can change across the width of the gel and lanes and bands can mix with each other. The sources of such inaccuracies stem from the lack of uniformity and homogeneity of the gel bed, electroendosmosis, thermal gradient and diffusion effects, as well as host of other factors. Inaccuracies of this sort are well known in the GEP art and can lead to serious distortions and inaccuracies in the display of the separation results. In addition, the band display data obtained from GEP separations is not quantitative or accurate because of the uncertainties related to the shape and integrity of the bands. True quantitation of linear band array displays produced by GEP separations cannot be achieved, even when the linear band arrays are scanned with a detector and the resulting data is integrated, because the linear band arrays are scanned only across the center of the bands. Since the detector only sees a small portion of any given band and the bands are not uniform, the results produced by the scanning method are not accurate and can even be misleading.
DNA molecules are polymers comprising sub-units called deoxynucleotides. The four deoxynucleotides found in DNA comprise a common cyclic sugar, deoxyribose, which is covalently bonded to any of the four bases, adenine (a purine), guanine(a purine), cytosine (a pyrimidine), and thymine (a pyrimidine), hereinbelow referred to as A, G, C, and T respectively. A phosphate group links a 3′-hydroxyl of one deoxynucleotide with the 5′-hydroxyl of another deoxynucleotide to form a polymeric chain. In double stranded DNA, two strands are held together in a helical structure by hydrogen bonds between, what are called, complimentary bases. The complimentarity of bases is determined by their chemical structures. In double stranded DNA, each A pairs with a T and each G pairs with a C, i.e., a purine pairs with a pyrimidine. Ideally, DNA is replicated in exact copies by DNA polymerases during cell division in the human body or in other living organisms. DNA strands can also be replicated in vitro by means of the Polymerase Chain Reaction (PCR).
Sometimes, exact replication fails and an incorrect base pairing occurs, which after further replication of the new strand results in double stranded DNA offspring containing a heritable difference in the base sequence from that of the parent. Such heritable changes in base pair sequence are called mutations.
In the present invention, double stranded DNA is referred to as a duplex. When the base sequence of one strand is entirely complimentary to the base sequence of the other strand, the duplex is called a homoduplex. When a duplex contains at least one base pair which is not complimentary, the duplex is called a heteroduplex. A heteroduplex duplex is formed during DNA replication when an error is made by a DNA polymerase enzyme and a non-complimentary base is added to a polynucleotide chain being replicated. Further replications of a heteroduplex will, ideally, produce homoduplexes which are heterozygous, i.e., these homoduplexes will have a complimentary, but altered sequence compared to the original parent DNA strand. When the parent DNA has the sequence which predominates in a natural population it is generally called the “wild type.”
Many different types of DNA mutations are known. Examples of DNA mutations include, but are not limited to, “point mutation” or “single base pair mutations” wherein an incorrect base pairing occurs. The most common point mutations comprise “transitions” wherein one purine or pyrimidine base is replaced for another and “transversions” wherein a purine is substituted for a pyrimidine (and visa versa). Point mutations also comprise mutations wherein a base is added or deleted from a DNA chain. Such “insertions” or “deletions” are also known as “frameshift mutations”. Although they occur with less frequency than point mutations, larger mutations affecting multiple base pairs can also occur and may be important. A more detailed discussion of mutations can be found in U.S. Pat. No. 5,459,039 to Modrich (1995), and U.S. Pat. No. 5,698,400 to Cotton (1997). These references and the references contained therein are incorporated in their entireties herein.
The sequence of base pairs in DNA codes for the production of proteins. In particular, a DNA sequence in the exon portion of a DNA chain codes for a corresponding amino acid sequence in a protein. Therefore, a mutation in a DNA sequence may result in an alteration in the amino acid sequence of a protein. Such an alteration in the amino acid sequence may be completely benign or may inactivate a protein or alter its function to be life threatening or fatal. On the other hand, mutations in an intron portion of a DNA chain would not be expected to have a biologic
Gjerde Douglas T.
Taylor Paul D.
Marschel Ardin H.
Transgenomic Inc.
Walker William B.
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