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Long distance polymerase chain reaction-based assay for detecting chromosomal rearrangements

a polymerase chain reaction and assay technology, applied in the field of coagulation, can solve the problems of common unamplification, and difficult optimization of primer concentration

Inactive Publication Date: 2009-04-02
CITY OF HOPE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011]Other aspects of the invention are specifically directed to determining the presence of an inversion in the factor VIII gene which causes hemophilia A. These methods comprise performing long distance PCR with 2 primers, 3 primers, 4 primers, or more than 4 primers. These methods allow one to detect the presence of males who have the inversion and therefore have hemophilia A and these methods allow one to determine whether females are carriers of the inversion.

Problems solved by technology

The inversions disrupt the factor VIII gene and cause almost half of all cases of severe hemophilia A. They are detected routinely by time-consuming and expensive Southern blots using a probe from Int22h1.
Multiplex PCR is a rapid and convenient method, but uneven amplification is common (Chamberlain et al., 1998).
Since primer concentration is often difficult to optimize, Shuber et al.

Method used

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  • Long distance polymerase chain reaction-based assay for detecting chromosomal rearrangements
  • Long distance polymerase chain reaction-based assay for detecting chromosomal rearrangements
  • Long distance polymerase chain reaction-based assay for detecting chromosomal rearrangements

Examples

Experimental program
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Effect test

example 1

Sequence for Flanking Regions of Factor VIII Gene Int22h1, Int22h2 and Int22h3

[0059]Fifty bp of 5′ flanking sequence of Int22h1 in intron 22 was available from GenBank (Accession Nos. X86011 and X86012). Fifty bp of flanking sequences were also known at both 5′ and 3′ ends of Int22h2 and Int22h3 (Naylor et al., 1995). Inverse-PCR (Ochman et al., 1988) was applied to amplify the unknown flanking sequences.

[0060]To obtain sequence for 5′ flanking Int22h1, a 6 kb segment was amplified with the Expand™ Long Template PCR system (Boehringer Mannheim). The primers used were: F8(E22)(89)32D: 5′-TGCCCGTCAGAAGTTCTCCAGCCTCTACATCT-3′ (SEQ ID NO:1) (bases 89-120 of Genbank Accession No. M88644) which is a primer corresponding to exon 22 of the factor VIII gene and Int22h(365)32U: 5′-GGTCAAGACTGAAATTAGCGTGTTAGGCAAGA-3′ (SEQ ID NO:2) (bases 384-415 of GenBank Accession No. X86011) which corresponds to the 5′ region of Int22h1. An ABI Model 377 sequencer was utilized to obtain 1319 bp of the 5′ fla...

example 2

Primer Design for the PCR Assay

[0063]Four primers are used in a multiplex PCR assay to detect the inversion in the factor VIII gene. These primers are labeled P, Q, A and B and their locations are indicated in FIG. 3A. These primers were designed with the aid of Oligo 5 software (National Biosciences). The Tm value of each PCR segment was estimated by the formula of Wetmur: Tmproduct=81.5+16.6 log [K+]+0.41% (G+C)−675 / length (Wetmur, 1991). The G+C content of the AB segment is 51% on average and the Tm is 81° C. The Tm values of the primers were estimated by the nearest neighbor method at 50 mM KCl and 250 μM DNA with the formula: Tmprimer=ΔH / {ΔS+R×ln(C / 4)}+16.6 log [K+]−273.15 (Breslauer et al., 1986; Freier et al., 1986). Each primer was designed to have a Tm that was 10° C. lower than the average Tm of the PCR products (73° C.). Primer lengths varied from 36-40 nucleotides. For primers P, Q and B, a high GC tail of two to five nucleotides was added in order to achieve Tm value of...

example 3

The PCR Assay

[0065]The PCR was performed from human genomic DNA isolated from white blood cells, although DNA from other cells could also be used. Unless otherwise stated, the PCR mixtures contained a total volume of 25 μL: 50 mM Tris-HCl, pH 9.2, 2.25 mM MgCl2, 16 mM (NH4)2SO4, 7.5% DMSO, 500 μM of dGTP and deaza-dGTP (62.5%:37.5% or 50%:50%), 500 μM of each of the other dNTPs, 250 ng of genomic DNA.

[0066]Three types of cycling conditions were utilized: three-temperature PCR, two-temperature PCR and S-PCR. The cycling conditions for three-temperature PCR were 94° C. for 12 seconds, 65° C. for 30 seconds and 68° C. for 14 minutes for the first 10 cycles (Perkin Elmer GeneAmp PCR System 9600). The remaining 20 cycles were performed by adding an extra 20 seconds to the elongation per cycle.

[0067]Conditions for two temperature cycling were: 94° C. for 12 seconds and 65° C. for 15 minutes for the first 10 cycles, with an extra 20 seconds added to the elongation per cycle for the remaini...

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Abstract

Methods are presented for determining the presence of an inversion in the factor VIII gene which cause hemophilia A. The methods encompass long distance, multiplex PCR (including overlapping PCR). The use of deaza-dGTP, high levels of DNA polymerases and high levels of DMSO aid in successfully performing the PCR. The use of a novel technique called subcycling PCR can also be applied as part of the methods. The technique allows for the determination of whether a person is homozygous or hemizygous for the inversion and has hemophilia A or whether a person is heterozygous for the inversion and is a carrier. The technique of long distance, multiplex PCR including use of deaza-dGTP, high levels of DNA polymerases and high levels of DMSO are applicable to the determination of the presence of other gross chromosomal aberrations such as deletions / inversions, translocations and inversions. The use of subcycling PCR can achieve efficient and more even amplification than normal two or three temperature PCR and is applicable to long distance, multiplex PCR.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]The present invention is a continuation-in-part of application Ser. No. 09 / 103,505 filed 24 Jun. 1998.BACKGROUND OF THE INVENTION[0002]Hemophilia A is one of the most common coagulation disorders with an incidence of about one in 5,000 males. The disease is caused by mutations in the factor VIII gene located on the X chromosome. About half the families with severe disease have a large genomic inversion of the factor VIII gene which separates the first 22 exons from the final four exons. This inversion results from a hotspot of recombination between a 9.5 kb region in intron 22 (Int22h1) and either of two extragenic, distal homologs, Int22h2 and Int22h3 near the Xq telomere which are repeats of Int22h1. These repeated sequences are more than 99% identical with one another (Naylor et al., 1995). Int22h2 and Int22h3 are in the opposite orientation of Int22h1 and therefore recombination produces an inversion. Intrachromosomal homologous recom...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q1/6883C12Q2600/16C12Q2600/156C12Q2527/149C12Q2527/125C12Q2527/101
Inventor LIU, QIANGSOMMER, STEVE S.
Owner CITY OF HOPE
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