Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-03-14
2003-12-02
Whisenant, Ethan (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024300
Reexamination Certificate
active
06656686
ABSTRACT:
TABLE OF CONTENTS
1. FIELD OF THE INVENTION
2. BACKGROUND OF THE INVENTION
1. METHODS BASED ON CHANGES OF ELECTROPHORETIC MOBILITY
2. MISMATCH CLEAVAGE METHODS
3. MISMATCH RECOGNITION METHODS
4. SEQUENCING METHODS
3. SUMMARY OF THE INVENTION
4. BRIEF DESCRIPTION OF THE FIGURES
5. DETAILED DESCRIPTION OF THE INVENTION
Potential applications of SDBC method
a. The construction of a cDNA library from cancer cells in lambda ZAP II vector and the generation of the single stranded (ss) phagemid library
b. Hybridization of ss cDNA library to normal poly (A)
+
RNA and the conversion into double strand plasmid DNA
c. Opening the loops from RNA-DNA duplexes
d. Ligation to stuffer and screening for mutated genes
e. Determination of the base changes site
6. EXAMPLES
6.1 Simultaneous Detection of Base Changes
6.2 Identification of cancer associated SNPs
a. The preparation of the probes for genes carrying base substitutions
b. Identification of the subpool of common SNPs
1. FIELD OF THE INVENTION
The present invention relates to methods for detecting polymorphisms in a nucleic acid sample without knowing the nucleotide sequence near the polymorphism. The invention further relates to a method for identifying polymorphisms that are specific for a particular tumor type.
2. BACKGROUND OF THE INVENTION
During the past few decades it has become clear that many human diseases are caused by alterations of cellular proteins, the molecules responsible for carrying out almost all cellular processes. Frequently, these alterations are the consequence of base changes in the nucleotide sequence encoding these cellular proteins. There are two common types of alterations: polymorphisms and mutations.
In humans, it is estimated that there is one base variation every 1-2 kb of homologous chromosomal sequence of any two individuals (Cooper et al. 1985; Kwok et al. 1994). Most of these variations, or polymorphisms, occur in noncoding sequence of the genome and do not alter the observed phenotype. However, some of them occur in the coding sequence and may result in the changes of the biological activity of the proteins they encode. The majority of these polymorphisms are compensated for by the organism, but some of them may lead to disease.
Mutations, however, are molecular alterations which may lead to abnormal protein function which can have serious consequences for the organism. Genetic mutations may be inherited or acquired during an organism's lifetime. Mutations may accumulate spontaneously or are induced by a variety of environmental physical and chemical agents. Organisms are subjected to nearly constant aggression by environmental agents. To prevent a rapid accumulation of multiple mutations and possible cell death, cells rely on DNA repair mechanisms able to correct these environmentally induced mutations. Occasionally, the repair mechanisms fail, and mutations persist leading to cell damage and/or death. If the accumulated mutations are not lethal, they may be transmitted to daughter cells causing genetic disorders in that or future generations.
Cancer may result from accumulation of mutations in multiple genes in one cell or its descendants. Often these mutations lead to the loss of ability to differentiate, to undergo apoptosis or to contact inhibition. The mutations may also lead to uncontrolled proliferation, the enhanced ability for invading surrounding tissues or for metastasizing.
More recently, as the risks for developing different diseases are extensively studied, it appears that variations in the structure of molecules responsible for a cell's handling and response to various agents may determine the propensity of an individual organism to develop a certain disease. The particular handling of an environmental agent will determine ultimately whether or not the agent will have any damaging effect on cell molecules. Thus the individual molecular profile at the level of DNA, RNA and cellular proteins may hold the key to understanding both the risk and the capacity of an individual organism for developing various diseases including cancer.
The determination of this individual molecular profile, that is the subtle differences in the structure of cellular molecules between organisms of the same species, is a task of considerable magnitude. Although there is a large amount of information accumulated during the past few decades regarding various mutations and polymorphisms in numerous genes, the progress towards achieving this objective is rather slow considering the magnitude of the task. This progress is dependent of our ability to detect efficiently and accurately base changes in most and perhaps in all cellular genes.
The existing methods for detection of base changes (Myers et al. 1998) can be broadly divided in the following groups:
1. Methods Based on Changes of Electrophoretic Mobility:
restriction enzyme finger printing (REF)
denaturing gradient gel electrophoresis (DGGE)
constant denaturing gel electrophoresis (CDGE)
carbodiimide mismatch detection
nondenaturing gel mismatch detection
single stranded conformational polymorphism (SSCP)
Restriction endonuclease fingerprinting (REF) (Liu and Sommers, 1995) is based on the change of the recognition site for restriction enzymes due a base alteration. This is detected as a change in the size of the DNA fragments generated by the endonuclease when the test and reference DNA are analyzed in parallel in a Southern blot for restriction fragment length polymorphism (RFLP).
Some methods are based on the change of the melting pattern of various DNA domains during the transition from single to double stranded form due to the presence of a base substitution in the sequence. The change in the melting pattern of a DNA fragment can be detected by electrophoresis in denaturing gradient gels (DGGE; Fisher and Lerman 1983), in denaturing constant gels (CDGE; Boresen et al. 1991) or by denaturing high performance liquid chromatography (dHCPL; Oefner unpublished). Using heteroduplexes between mutated and wild type DNA sequences one can also detect base changes by electrophoresis in nondenaturing gels (White et al. 1992). The change in mobility can be enhanced by the attachment at the site of mismatched bases, of a chemical moiety such as carbodiimide (Novack et al. 1986).
Other methods rely on the change of the electrophoretic mobility in nondenaturing gels of a single stranded DNA fragment due to the presence of a base change in the sequence (SSCP; Orita et al. 1989). At present single stranded conformational polymorphism (SSCP) is the most widely used method for detection of base changes. It implies the amplification by RT-PCR of 200-300 bp gene fragment containing the base alteration. The PCR fragment is then subjected to electrophoresis in a nondenaturing polyacrylamide gel including 5-10% glycerol, along with the homologous fragment from the wild type gene. A difference in mobility between the two fragments indicates the presence of a mutation in the target fragment which is then confirmed by sequencing. This method requires the knowledge of the nucleotide sequence of the mutated gene and the approximate location of the mutation so that appropriate PCR primers could be selected for amplification of the target fragment.
2. Mismatch Cleavage Methods:
RNase A cleavage
chemical cleavage of mismatches (CCM)
bacteriophage T4 endonuclease 7 cleavage at mismatches (ECM)
mismatch repair enzyme cleavage with
E. coli
Mut Y protein (MREC)
These methods are based on the cleavage of the single stranded RNA or DNA at the site of mismatched bases in heteroduplexes formed between the complementary strands from a reference and a test sample. The method of cleavage could be the digestion with RNase A (Myers et al. 1985), a chemical reaction (CCM; Cotton et al. 1988), or digestion with bacteriophage T4 endonuclease VII (ECM; Youil et al. 1995) or with
E. coli
MutY protein (MREC; Lu and Hsu 1991).
3. Mismatch Recognition Methods:
mismatch repair detection (MRD)
oligonucleotide array hybridization method (“DNA chips”)
Mismatch repair detection (MRD, Faham and
Pennie & Edmonds LLP
Whisenant Ethan
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