Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
1999-10-06
2001-09-11
Warden, Jill (Department: 1743)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S466000
Reexamination Certificate
active
06287441
ABSTRACT:
BACKGROUND OF THE INVENTION
Single-strand conformation polymorphism (SSCP) is the most widely used method for mutation scanning. With SSCP, single-base sequence changes can be detected by altered electrophoretic migration of one or both single strands on a non-denaturing gel. SSCP does not detect all sequence changes with one electrophoresis condition and its sensitivity is a complex function of sequence context and size (Glavac and Dean, 1993; Hayashi and Yandell, 1993; Hongyo et al., 1993; Liu and Sonuner, 1994; Michaud et al., 1992; Sarkar et al., 1992a; Sarkar et al. 1992b; Sheffield et al., 1993; Takahashi-Fujii et al., 1993).
Previous work suggested that the idiosyncratic nature of SSCP sensitivity is a function of both the distribution of mobility of single-base changes and the mobility of the wild type sequences relative to that of all single-base changes (see FIG.
1
). For a 200 bp segment, there are 600 possible variants that differ by a single-base substitution. If it were possible to generate all 600 possible variants and to plot the mobility in units of band widths, it is apparent that the sensitivity of SSCP will be less for a segment in which the mobility of the wild type sequence is close to the mode (see FIG.
1
A). If the variance of mobility is wider (FIG.
1
B), SSCP sensitivity, on average, will be higher than with the first condition. However, this is not necessarily so, because the location of the wild type sequence within the distribution is also critical.
At least two ways to increase the sensitivity of SSCP have been described. In one approach, SSCP is hybridized with another method in order to generate the redundancy of mutation-containing segments necessary to detect virtually all mutations. For example, in dideoxy fingerprinting (ddF), SSCP is combined with Sanger dideoxy sequencing (Lin and Sommer, 1994; Sarkar et al., 1992b). SSCP can also be combined with restriction endonuclease fingerprinting (REF) (Liu and Sommer, 1995) and with bi-directional dideoxy fingerprinting (bi-ddF) (Liu et al., 1996). A Sanger dideoxy termination reaction is performed with one dideoxy terminator. The terminated single-stranded segments are electrophoresed through a non-denaturing gel. The ladder of segments subsequent to the mutation contain the same mutation with different 3′ ends. In a second approach, SSCP is performed under two or more conditions. Typically, two temperatures are utilized, and occasionally, two temperatures with and without glycerol (Glavac and Dean, 1993; Liu and Sommer, 1994).
Recently, we reported that the pattern of SSCP shifts varied markedly when HEPES was added to standard TBE buffer (Liu and Sommer, 1998). The correlation coefficient (R) between these two conditions was 0.46. These results hint that sugar/base and sugar/sugar interactions are more important than secondary structure, which should not be much affected by the addition of HEPES.
Herein, we report a detailed analysis of gel matrix, running buffer, temperature, and additive to search for a set of sensitive and complementary electrophoretic conditions for SSCP analysis. ddF was utilized in order to provide a very large sample of mutation-containing segments for analysis. From the data, five conditions were chosen and SSCP
5
analysis was performed with 100% sensitivity in two blinded analyses.
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended List of References.
SUMMARY OF THE INVENTION
Experiments were performed to test for a set of SSCP conditions that would detect virtually all mutations in a nucleic acid being analyzed. The effects of buffer, gel matrix, temperature, and additive were all tested. Dideoxy fingerprinting was used as a tool to generate a large statistical sample (about 1,500) of mutation-containing single-stranded segments in order to evaluate adequately the sensitivity under a given condition. Mutations in exons H and B/C of the factor IX gene were utilized. SSCP sensitivity, as conveniently measured by the average SSCP efficiency, varied with conditions. Correlation coefficients (R) identified pairs of conditions that were either close to independent or complementary. Five conditions were selected with sufficient redundancy to detect all the mutations in the set tested. The sensitivity of multi-conditional SSCP (SSCP
5
) was determined by blinded analyses on samples containing mutations in all the eight exon regions in the factor IX gene. 2.5 kb of factor IX gene sequence were scanned in one lane by 15 PCR-amplified segments (125 kb of sequence scanned per gel). All of the 84 single-base substitutions were detected in the blinded analyses, the first consisting of 50 hemizygous mutant and wild type samples and the second consisting of 50 heterozygous mutant and wild type samples. 90% of these mutations were detected by more than one condition. SSCP
5
is estimated to be five times faster than fluorescent DNA sequencing for the detection of virtually all mutations when the five conditions are applied.
REFERENCES:
Schneider-Stock et al. (“Improved detection of P53 mutations in soft tissue tumors using new gel composition for automated nonradioactive analysis of single-strand conformation polymorphism”, Electrophoresis (1997), 18(15), 2849-2851).*
Rubio et al. (Differentiation of citrus tristeza closterovirus (CTV) isolates by single-strand conformation polymorphism of the coat protein gene, Ann. Appl. biol. (1996), 12993), 479-489).*
Vasquez et al. (detection of polymorphism in the Trypanosoma cruzi tcP2.beta. gene family by single strand conformational analysis (SSCA)), Gene (1996), 180(1/2), 43-48.*
Birren, B.W. et al. “Optimized conditions for pulsed field gel electrophoretic separations of DNA”,Nucleic Acids Research(1988); 16(15):7563-7582, Month unknown.
Johnson, P.H. and Grossman, L.I. “Electrophoresis of DNA in Agarose Gels. Optimizing Separations of Conformational Isomers of Double-and Single-Stranded DNAs”Biochemistry(1977); 16(19):4217-4225, Month unknown.
Landick, R. et al. “Optimization of Polyacrylamide Gel Electrophoresis Conditions Used for Sequencing Mixed Oligodeoxyribonucleotides”DNA(1984); 3(5):413-419, Month unknown.
Oto, M. et al. “Optimization of Nonradioisotopic Single Strand Conformation Polymorphism Analysis with a Conventional Minislab Gel Electrophoresis Apparatus”,Analytical Biochemistry(1993); 213:19-22, Month unknown.
Raghava, G.P.S. “DNAOPT: A Computer Program to Aid Optimization of DNA Gel Electrophoresis and SDS-PAGE”,Bio-Techniques(1995); 18(2):247-278, 280 (p. 279 is advertisment).
Velleman, S.G. “A Method for Empirically Optimizing the Detection of DNA Polymorphisms in Genomic DNA by Denaturing Gradient Gel Electrophoresis”,Bio Techniques(1992); 12(4):521-524.
Kukita, Y., et al. “SSCP Analysis of Long DNA Fragments in Low pH Gel”, Month unknownHuman Mutation, 1997; 10:400-407.
Liu, Q., et al. “Bi-directional dideoxy fingerprinting (Bi-ddf): a rapid method for quantitative detection of mutations in genomic regions of 300-600 bp”,Human Molecular Genetics, Month unknown 1996; 5(1):107-114.
Liu, Q. and Sommer, S. “The SSCP Phenomenon: Addition of HEPES Buffer Dramatically Affects Electrophoretic Mobility”,Bio Techniques, Jul. 1998; 25(1):50-56 (4 pages).
Sasaki, T. et al. “ATM Mutations in Patients With Ataxia Telangiectasia Screened by a Hierarchical Strategy”,Human Mutation, Month unknown 1998; 12:186-195.
Liu Qiang
Sommer Steve S.
City of Hope
Noguerola Alex
Rothwell Figg Ernst & Manbeck
Warden Jill
LandOfFree
Multi-conditional SSCP (SSCP5): a rapid method for mutation... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Multi-conditional SSCP (SSCP5): a rapid method for mutation..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Multi-conditional SSCP (SSCP5): a rapid method for mutation... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2522437