Multiple PCR method based on selective probe and application thereof

A selectivity and probe technology, applied in multiplex PCR methods and its application fields, can solve problems such as amplification of multiple DNA sequences, difficult conditions to control, primer-primer dimer aggregation, etc., to enhance design sensitivity and simplicity Effect

Inactive Publication Date: 2007-11-07
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Abstract
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  • Claims
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Problems solved by technology

Since CpG islands are CG-rich sequences, when analyzing DNA methylation based on MSRE, it is difficult to amplify multiple DNA sequences at the same time, even with multiplex PCR amplification based on padlock probes or molecular inversion probes. The large number of CpG sites in CpG leads to the existence of multiple MSRE sites. Therefore, according to the design scheme of classic padlock probes or molecular inversion probes, it is difficult to obtain probes that meet the requirements.
In addition, for the bisulfite-modified sequence, since the non-methylated cytosine has been converted into urac...

Method used

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  • Multiple PCR method based on selective probe and application thereof
  • Multiple PCR method based on selective probe and application thereof
  • Multiple PCR method based on selective probe and application thereof

Examples

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Effect test

Embodiment 1

[0058] (1) Construction of selective probes

[0059] Fig. 1 is a schematic diagram of the structure of several selective probes of the present invention, as can be seen from Fig. 1, the selective probe provided by the present invention comprises at least four partitions, and it is characterized in that the middle part of the selective probe is two universal sequence regions ( P1 and P2 regions in Figure 1), the 5'- and 3'-ends of the selective probe are respectively the same nucleic acid single strand of the target double-stranded nucleic acid or the 5'- and 3'-ends of the target single-stranded nucleic acid. Two target nucleic acid complementary regions (C1, C2 districts in Fig. 1) that are complementary to each other, and, when the starting nucleic acid template is RNA or mRNA, the target nucleic acid complementary region of the 3'-end of the selective probe can be Olig d( T) (Oligo d(T) region in Figure 1), the length of Oligo d(T) is 8-40mer. There can also be a uracil re...

Embodiment 2

[0090] This example provides a multiplex PCR method for simultaneously detecting two CpG island sequences in the promoter region of the p16 gene.

[0091] (1) Design selective probes

[0092] Selective probe P16-V1 targets Chr9: 21,964,579-21,965,306 (728bp) CpG island sequence, its sequence is as follows:

[0093] 5′P-CATTCGCTAAGTGCTCGGAGCAGAATCGTCCAGTCGCAGT GAGG CATGTACCGTCGTTGT AGTCCTCCTTCCTTGCCAAC-3′

[0094] Wherein, the 5'- and 3'-end bold sequences are target nucleic acid complementary regions, and the 5'-terminal target nucleic acid complementary region is complementary to the 21965049-21965068 sequence of chromosome No. 9 (region C1 in Figure 1), and the 3'- The end-target nucleic acid complementary region is complementary to the sequence of 21965144-21965163 of chromosome 9 (region C2 in Figure 1); italicized (region P1 in Figure 1) and underlined sequence (region P2 in Figure 1) is a general sequence region . The 5'- and 3'-terminal bases of the selective prob...

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Abstract

The present invention relates to nucleic acid amplification, and is especially multiple PCR process based on selective probe for simultaneous amplification of several nucleic acid segments and its application. The multiple PCR process includes the following steps: 1. constructing selective probe; 2. extension-coupling reaction; 3. PCR amplification; and 4. detecting PCR amplification product. The present invention features the selective probe and the PCR amplification product with difference in length. The process has greatly increased analysis flux, saving in the nucleic acid template consumption for detecting the sample, high compatibility of the detection system and other advantages.

Description

technical field [0001] The invention relates to nucleic acid amplification, in particular to a selective probe-based multiple PCR method and its application. Background technique [0002] Multiplex PCR (Polymerase Chain Reaction) technology is a technology for simultaneously amplifying multiple target nucleic acids in the same reaction tube, among which the most typical representative is a series of gene analysis technologies developed based on padlock probes. The classic padlock probe is composed of three parts, wherein two parts of the sequence are oligonucleotide fragments at both ends of the padlock probe, the two parts of the oligonucleotide fragments are complementary to the target nucleic acid, and the third part of the oligonucleotide fragment Some oligonucleotide fragments are connected into a whole. When the padlock probe forms a hybrid with the target nucleic acid, there is no space between the 5'- and 3'-ends of the padlock probe, and the two are closely adjacent...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12P19/34
Inventor 黄庆府伟灵王珏黄君富
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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