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A method of DNA amplification

A nucleic acid, region-based technology for use in the field of amplification of nucleic acid regions of interest

Inactive Publication Date: 2010-09-29
MONOQUANT PTY LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the bottleneck during PCR is worse for undesired amplicons generated from sequences that can be primed with primer 2 as forward and reverse primers

Method used

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  • A method of DNA amplification
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  • A method of DNA amplification

Examples

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Effect test

Embodiment 1

[0247] BCR-ABL DNA Breakpoint Amplification Using Bottleneck PCR

[0248] BCR-ABL DNA breakpoints were amplified in 4 rounds of PCR screening with BCR- and ABL-specific primers. Six BCR-specific primers and 282 ABL-specific primers were designed to span the major breakpoint regions of BCR (3.2kb) and ABL (140kb) DNA, respectively.

[0249] The first round of PCR amplification was performed in 25 μl containing 50 ng of a single BCR-specific primer, 100 ng of all 282 ABL-specific primers (350 pg each), 50 ng Tag A, 50 ng of genomic DNA, 50 mM KCl, 2 mM Tris HCl (pH8.4), 1 U Platinum Taq DNA polymerase (Invitrogen), 5 mM MgCl 2 and 300 μM each of dUTP, dATP, dGTP and dTTP. The amplification conditions are: 95°C for 4 minutes; then 6 cycles of 97°C for 1 minute, 65°C for 1 minute, every 2 cycles the temperature is lowered by 1°C, and 72°C for 1 minute; then 4 cycles of 96°C for 30 seconds, 62°C for 1 minute, after the first 2 cycles, the temperature is lowered by 1°C and 72°C f...

Embodiment 2

[0253] Amplification of PML-RARα DNA Breakpoints by Bottleneck PCR

[0254] Isolation of PML-RARα translocation breakpoints from patients with acute promyelocytic leukemia by bottleneck PCR. Patient DNA was amplified with multiple RARα primers and a single PML primer, followed by 2 rounds of bottleneck PCR. The amplified DNA was electrophoresed on a 2% agarose gel ( Figure 9 a). To confirm that the breakpoint was isolated, RARα and PML primers spanning the breakpoint were designed using the breakpoint sequence, and patient DNA was amplified with these primers for one round ( Figure 9 b). also confirmed Figure 9 a The sequence of the amplified band shown on the gel ( Figure 9 c).

Embodiment 3

[0256] Gene Walking Using Degenerate Primers and Bottleneck PCR

[0257] Gene walking along the 3 genes APC, BRCA1 and myocillin was performed with 50 ng of gene-specific forward primer, 50 ng of one of various degenerate reverse primers and 50 ng of reverse marker primer. The degenerate reverse primer has 4-6 random normal residues at the 3' end, followed by 3-6 degenerate residues, followed by a random marker sequence of 12-18, usually 18 normal residues. The most commonly used degenerate primer has 5 fixed bases at the 3' end, followed by 5 degenerate bases, followed by an 18 base marker sequence (5'TGCTAGGATCCAAGGNNNNNATTCG3' (SEQ ID NO: 1)). The reverse marker primer has the same sequence as the marker on the degenerate reverse primer. 5 PCR cycles (annealing at 35°C for 5 minutes), followed by 15 cycles (annealing at 55°C for 3 minutes).

[0258] PCR performed in 25 μl volume with 50 ng total DNA, 5 mM MgCl 2 , 0.1 mM dUTP, 0.2 mM dTTP and 0.3 mM each of dCTP, dATP an...

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Abstract

The present invention relates generally to a method of amplifying a nucleic acid region of interest and, more particularly, to a method of amplifying a nucleic acid region of interest using a PCR method designed to minimise the generation of amplicons from primers which have bound to nucleic acid regions other than the specific region of interest. The method of the present invention is based on the determination that by rendering inefficient the functionality of either the forward primer or the reverse primer, the rate of amplification of irrelevant nucleic acid regions can be reduced relative to amplification of the region of interest. The provision of a selective means of amplifying a nucleic acid region of interest is useful in a range of applications including, but not limited to, the diagnosis and / or monitoring of disease conditions which are characterised by specific gene sequences, the characterisation or analysis of gene regions of interest, the identification or characterisation of DNA breakpoint regions and the isolation of gene sequences of interest where only the nucleotide sequence at one end of the gene sequence of interest is known.

Description

technical field [0001] The present invention relates generally to methods for amplifying a nucleic acid region of interest, and more particularly to methods for amplifying a nucleic acid region of interest using a PCR method designed for use in the Amplicon generation of primers for nucleic acid regions is minimized. The method of the invention is based on the determination that by making the functionality of the forward or reverse primers inefficient, the rate of amplification of unrelated nucleic acid regions can be reduced relative to the amplification of regions of interest. Means that provide selective amplification of nucleic acid regions of interest may have a range of applications including, but not limited to, diagnosis and / or monitoring of disease conditions characterized by specific gene sequences, identification or analysis of gene regions of interest, DNA breakpoints Identification or characterization of a region, isolation of a gene sequence of interest where on...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6848C12Q2527/143C12Q2527/107C12Q2525/155
Inventor A·A·莫利
Owner MONOQUANT PTY LTD
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