Multiple-fluorescent PCR (polymerase chain reaction) amplification reagent and kit for detecting instability of microsatellite

An unstable, multiple fluorescence technology, applied in recombinant DNA technology, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problem of easy cross-overlapping of fluorescence peaks

Active Publication Date: 2017-09-29
常州桐树生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, after PCR amplification of microsatellite loci (BAT25, BAT26, D2S123, D5S346, and D17S250) with traditional PCR amplification reagents, the fluorescence peaks of the amplified products obtained from each microsatellite locus are easily crossed after electrophoresis detection. Overlapping, so it is difficult to simultaneously detect changes in microsatellite loci BAT25, BAT26, D2S123, D5S346 and D17S250 in the same PCR system

Method used

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  • Multiple-fluorescent PCR (polymerase chain reaction) amplification reagent and kit for detecting instability of microsatellite
  • Multiple-fluorescent PCR (polymerase chain reaction) amplification reagent and kit for detecting instability of microsatellite
  • Multiple-fluorescent PCR (polymerase chain reaction) amplification reagent and kit for detecting instability of microsatellite

Examples

Experimental program
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Effect test

Embodiment 1

[0077] Check the gene number of the microsatellite site to be tested in the Gene Bank, and determine the main repeat sequence of each microsatellite site and the base sequence of about 1000 bp upstream and downstream of the main repeat sequence. The relevant information of the microsatellite site is shown in Table 1 shown.

[0078] Table 1: Basic information of microsatellite loci

[0079] Microsatellite loci

gene number

major repeat sequence

BAT25

L04143

(A) 25

BAT26

U41210

(A) 26

D2S123

Z16551.1

(CA) 15

D5S346

NM_005669

(CA) 20

D17S250

NR_033753.2

(CA) 24

[0080] Targeted design of upstream primers and downstream primers for each microsatellite locus, and correspondingly labeled fluorescent groups, as shown in Table 2.

[0081] Table 2: Upstream and downstream primers for microsatellite loci

[0082]

[0083]

[0084] The designed primers were biosynthesized by Sangon,...

Embodiment 2

[0086]The upstream primer BAT25-F1, downstream primer BAT25-R1, upstream primer BAT26-F1, downstream primer BAT26-R1, upstream primer D2S123-F1, downstream Primer D2S123-R1, upstream primer D5S346-F1, downstream primer D5S346-R1, upstream primer D17S250-F1 and downstream primer D17S250-R1 are the same as in Example 1, the difference is that microsatellites for the control are also added in this example The upstream primer Penta C-F1 and the downstream primer Penta C-R1 designed at site Penta C. The relevant information of the microsatellite locus Penta C is shown in Table 3.

[0087] Table 3: Basic information of microsatellite locus Penta C

[0088] Microsatellite loci

gene number

major repeat sequence

Penta C

AL138752

(AAAAG) 3

[0089] The upstream primer Penta C-F1 and the downstream primer Penta C-R1 designed by Penta C were specifically designed for the microsatellite site Penta C, and correspondingly labeled fluorophores, as shown...

Embodiment 3

[0095] The upstream primer BAT25-F1, downstream primer BAT25-R1, upstream primer BAT26-F1, downstream primer BAT26-R1, upstream primer D2S123-F1, downstream Primer D2S123-R1, upstream primer D5S346-F1, downstream primer D5S346-R1, upstream primer D17S250-F1 and downstream primer D17S250-R1 are the same as in Example 1, the difference is that microsatellites for the control are also added in this example The upstream primer Penta D-F1 and the downstream primer Penta D-R1 designed at site Penta D. The relevant information of the microsatellite locus Penta C is shown in Table 5.

[0096] Table 5: Basic information of microsatellite loci Penta D

[0097] Microsatellite loci

gene number

major repeat sequence

Penta D

Ac000014

(AAAAG) 8

[0098] The upstream primer Penta D-F1 and the downstream primer Penta D-R1 designed by Penta D were specifically designed for the microsatellite site Penta D, and correspondingly labeled fluorescent groups, as...

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Abstract

The invention discloses a multiple-fluorescent PCR (polymerase chain reaction) amplification reagent and kit for detecting instability of a microsatellite. The PCR amplification reagent comprises upstream primers and downstream primers which are designed for microsatellite sites BAT25, BAT26, D2S123, D5S346 and D17S250, so that multiple microsatellite sites can be simultaneously amplified in a same PCR system, and the instability of the microsatellite is detected by the multiple-fluorescent PCR. The PCR amplification reagent has the advantages that after electrophoresis detection, the fluorescent peaks produced by the amplified products of the microsatellite sites are not crossed or superposed; the detection efficiency is high, and the sensitivity is good.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a multiple fluorescent PCR amplification reagent and kit for detecting microsatellite instability. Background technique [0002] Microsatellites, also known as Short tandem repeats (STRs) or Simple sequences repeats (SSRs), are widely distributed in the human genome. A repeat sequence generally consists of 1-6 nucleotides, especially a dinucleotide repeat sequence (CA / GT)n is the most common, where n is the number of repeats, usually 15-60 times. Microsatellites can specifically bind proteins or directly encode proteins, and some may be involved in the folding of chromatids or the formation of telomeres. Microsatellites play a role in gene regulation by changing DNA structure or binding to specific proteins. They are a new class of molecular markers with high polymorphic information capacity. [0003] Microsatellite instability (Microsatellite Instability, MSI) refers to the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q2537/143C12Q2563/107
Inventor 严令华韩宁宁袁青
Owner 常州桐树生物科技有限公司
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