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Semi-specific amplification primer group, method and kit for quickly detecting chromosome number abnormality

A technology for chromosome number and amplification primers, which is applied in the field of semi-specific amplification primer sets and kits for rapid detection of abnormal chromosome number, can solve the problems of unsatisfactory research and application, low throughput, and high cost. Achieve the effects of improved sequencing depth, short library construction time, and improved detection cycle

Active Publication Date: 2013-11-20
ANNOROAD GENE TECH BEIJING +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional first-generation sequencing method has many defects such as low throughput, long cycle time, and high cost, which can no longer meet the needs of research and application.

Method used

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  • Semi-specific amplification primer group, method and kit for quickly detecting chromosome number abnormality
  • Semi-specific amplification primer group, method and kit for quickly detecting chromosome number abnormality
  • Semi-specific amplification primer group, method and kit for quickly detecting chromosome number abnormality

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1 The acquisition of LINEs region primers

[0090] This patent designs a set of special primers, which can bind to a series of LINEs regions on the genome during PCR annealing. At the same time, it ensures the existence of certain specific SNP polymorphisms, which can be used to locate the relative position of an amplified fragment in the original genome.

[0091] Through the Repeat Library provided by www.repeatmasker.org, extract all LINEs regions, and use the BWA algorithm in the whole genome to search for fragments with a length of 130 bp-180 bp in the extracted LINEs region, which must meet the following conditions : The number of SNP sites in the fragment region includes 10-20. After subsequent manual screening one by one, we selected one of the regions as an example. And designed a pair of primers as follows:

[0092] forward primer 1a

[0093] 5'-GGCTATAAACGCAGGCCACTNNNNNNNNNNNNNNNNNGGGAGGGGAACATCACAC-3'

[0094] or forward primer 1b

[0095] 5'-GG...

Embodiment 2

[0108] Example 2 Detection method for abnormal number of chromosomes

[0109] The detection method of the present invention can be used for, but is not limited to, abnormal chromosome numbers such as trisomy 13, trisomy 18, and trisomy 21 in adults. It can also be used to non-invasively detect abnormal chromosome numbers in fetuses by analyzing the peripheral blood of pregnant women.

[0110] 1. Extraction of sample DNA:

[0111] In this embodiment, a total of 8 maternal blood samples were drawn (sample numbers are: S_042, S_053, S_055, S_058, S_064, S_066, S_071, S_078). 5 ml of peripheral blood was extracted from each sample, and after two high-speed centrifugation at 4°C, a plasma sample from which blood cells were removed was obtained. Plasma samples were extracted using a DNA extraction kit produced by Qiagen, or other conventional DNA extraction methods.

[0112] 2. Library construction:

[0113] Target fragment amplification: select forward primer 1b and revers...

Embodiment 3

[0132] Embodiment 3 Preparation of kit

[0133] The first round of specific amplification primers obtained in Example 1:

[0134] Forward primer 1a:

[0135] 5'-GGCTATAAACGCAGGCCACTNNNNNNNNNNNNNNNNNGGGAGGGGAACATCACAC-3'

[0136] or forward primer 1b:

[0137] 5'-GGCTATAAACGCAGGCCACTCBDVHBDVHBDVHBDVHBDVHGGGAGGGGAACATCACAC-3'

[0138] Reverse Primer 1:

[0139] 5'-TCTGAGGAATCAGCAATGCAGCGATCATGGTGGTTTGCTGCA-3'

[0140] The design structure of the primer pair is as follows Figure 6 shown.

[0141] Primers designed for sequencing library preparation obtained in Example 1:

[0142] Forward primer 2:

[0143] 5'-AATGATACGGCGACCACCGAGATCTACACGGCTATAAACGCAGGCCACTC-3'

[0144] Reverse primer 2:

[0145] 5'-CAAGCAGAAGACGGCATACGAGATNNNNNNNNTCTGAGGAATCAGCAATGCAGCGAT-3'

[0146] Wherein, B, D, V, H and N in the two sets of primer pairs are degenerate bases. NNNNNNNNN in reverse primer 2 is a specific base, which is the tag sequence of the sample, and the tag sequence is diffe...

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Abstract

The invention provides a semi-specific amplification primer group, a method and a kit for quickly detecting chromosome number abnormality. The invention relates to two groups of amplification primers. The kit provided by the invention consists of the following components: a 10*PCR buffer solution, ddH2O, a dNTP solution, PlatinumPf*DNA polymerase (2.5U / mu L), primer pairs of amplification primer 1 with concentration of 25 mu m / mu l, an amplification primer 2 and purified buffer (QIAGENMinElutePCRPurificationKit). Another object of the invention is to provide a method for quickly detecting chromosome number abnormality through the semi-specific amplification primer group. By adopting the invention, the chromosome number abnormality can be detected quickly.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to biotechnology and medicine, and specifically relates to a set of primers for semi-specific amplification of Long Interspersed Elements (LINE) in the genome, and a rapid construction of DNA with the primer set as the core. The invention relates to a method for sequencing a library, and also relates to a kit and a method for detecting an abnormal number of chromosomes. Background technique [0002] In the mid-1970s, Frederick Sanger invented the Sanger Dideoxy Chain Termination Method (Chain Termination Method) [1] , making it possible to directly detect DNA sequences. It uses a polymerase chain reaction (PCR) to complete the sequencing process. By randomly adding four dideoxyribonucleotides of ddATP, ddGTP, ddCTP and ddTTP, fragments of different lengths can be amplified by PCR, and the sequence of the original DNA sample can be detected by detecting the species of dideoxyribonucleoti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 陈重建梁峻彬刘洋洋
Owner ANNOROAD GENE TECH BEIJING
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