Multi-PCR detection method for minim DNA

A detection method and multiple technology, applied in the field of ultra-sensitive detection of trace DNA by multiple PCR technology, can solve problems such as inability to perform multi-site analysis, and achieve the effect of avoiding competition inhibition problems, wide application prospects, and simple operation process

Inactive Publication Date: 2008-08-20
DONGHUA UNIV +1
View PDF0 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The whole operation is simple and easy, and can effectively detect trace amount of template D

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Multi-PCR detection method for minim DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Studies on the methylation of GnRH neurons:

[0026] (1) Embed 500 mouse GnRH neuron cells with low-melting point agarose, and extract DNA;

[0027] (2) The extracted DNA is treated with sulfite, and about 90% of the DNA will be degraded in this step, so in fact the remaining DNA is quite small;

[0028] (3) Obtain DNA sequences of 10 candidate genes that may be methylated in GnRH neurons through GeneBank of NCBI, use software to design primers, and synthesize related sequences, see Table 1; and dilute these primers to 10p / mol.

[0029] Table 1 Primer Sequence

[0030] Primer name

Sequence (5'→3')

GnRHL-F

TTTGTTTTGTTTTGTTTTTGA

GnRHL-R

AACTTCAAAACTAAAAAAAAACTA

P2ry2L-F

TTGGTATTAGTAGTTTGAAAG

P2ry2L-R

ACCCTTCTAAACAAATACTTC

NmbrL-F

AAGTTTTAAAGATAGAAAAAAGGT

NmbrL-R

ATTAAAAAAAAACCTAAAAAACAT

[0031] GPR54L-F

GTTGTTGGGTTAGAAGTTTTGTTT

GPR54L-R

TCTCACAACAATAAT...

Embodiment 2

[0035] Whole-genome sequencing of the GPR54 gene in oral mucosal exfoliated cells:

[0036] (1) Collect oral cotton swabs from 20 patients with precocious puberty, and use a kit to extract DNA.

[0037] (2) The whole genome sequence of the GPR54 gene was obtained through NCBI's GeneBank, and the sequence analysis showed that the whole genome sequence of the GPR54 gene could be completely covered only by the amplification of 4 fragments. Use software to design primers and synthesize related sequences, see Table 2, and dilute each of these primers to 10p / mol.

[0038] Table 2 Primer Sequence

[0039] Primer name

Sequence (5'→3')

L-IF

GCG ACA GAG CGA GAT GCT GT

L-IR

TGG TGG ATT AGA GGC GTC AG

[0040] L-IIF

TGG TGA AAC CTG GTC TCT AC

L-IIR

GCA TCA GCG CCG CGA AGA AG

L-IIIF

AGC ACA GCT GCC CTC TGG AC

L-IIIR

CTG AGT GCA TCT CCC TCC GT

L-IVF

CTC AAC CCG CAC TGG ACA CT

...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to a multi-PCR detection method of trace DNA, which can avoid the competition inhibition problem due to the excessive primers of the multi-PCR technology by changing the concentration and the adding mode of the primers, so the multi-PCR detection method can be used for the very limited DNA of an initial sample, in particular to a plurality of very valuable clinical samples, such as, fertilized eggs and the obtained cells by laser capture and microdissection, etc. The whole detection proposal of the invention has simple operation process, high specificity, very high sensitivity and PCR efficiency, easy automation and wide application prospect in practical application.

Description

technical field [0001] The invention belongs to the field of nucleic acid detection, and in particular relates to a multiplex PCR technology which realizes supersensitive detection of trace DNA by changing the concentration of primers and the way of adding primers. Background technique [0002] Polymerase chain reaction (PCR) is the most commonly used amplification technology in biological detection today, and it can amplify a certain DNA fragment hundreds of thousands to millions of times within a few hours. And with the popularization and application of fluorescent quantitative PCR, the quantification of the target sequence can also be completed accurately. However, in general PCR, only one pair of primers is used, the throughput is low, and the initial template concentration is required, so the sensitivity is low. But for many studies, the DNA of the starting sample is very limited, especially some very precious clinical samples, such as fertilized eggs, cells obtained b...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12Q1/68
Inventor 王剑晖于明辉李凯陆炯肖君华
Owner DONGHUA UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap