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Amplification of target sequences

A technology of primer amplification and DNA sequence, which is applied in the field of target sequence amplification, and can solve the problems of the possibility of uneven sequence coverage, excessive or insufficient specific regions, etc.

Pending Publication Date: 2019-02-05
PERKINELMER HEALTH SCIENCES (AUSTRALIA) PTY LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, while whole genome amplification provides the ability to amplify genomic DNA for analysis, the amplification of specific target sequences from the amplified genomic DNA has several disadvantages, such as one or more of the following: allele drop out and / or bias events, potential for uneven sequence coverage, over- or under-representation of specific regions, reproducibility, and introduction of errors into that specific target sequence

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Examples

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Embodiment 1

[0355] Example 1-Whole genome amplification and gene targeting procedures

[0356] The following procedures are used for whole genome amplification (WGA) and gene targeting studies described herein.

[0357] 1. Materials and methods

[0358] The first round of whole genome amplification and gene targeting procedures:

[0359] (i) To 6.5μl PCR grade H 2 O add 1μl cell lyase (heat-resistant protease; EmbryoCellect TM Kit, RHS; number RHS1001); this is called cell lyase Dil 1.

[0360] (ii) Generate a cell lysis mixture by mixing the reagents in Table 1:

[0361] Table 1

[0362] Component

Volume for 1 cleavage reaction

PCR grade H 2 O

2.7

Cell lysis buffer

0.15

Cell Lyase Dil 1

0.15

total capacity

3

[0363] (iii) Add 3 μl of cell lysis mix to the cells or DNA tube. Tap the solution to the bottom of the tube or centrifuge briefly. Don't vortex.

[0364] In this regard, a single cell usually contains about 6 pg of DNA.

[0365] (iv) Incubate the sample in a thermal cycler with th...

Embodiment 2

[0402] Example 2-Using a single cell to verify the enrichment of targeted genes in WGA PCR

[0403] Research is conducted to validate the protocol for enriching target genes.

[0404] Add two sets of gene-specific primers (P) (0.5μM, 0.25μM or 0.1μM concentration) to WGA (multiplex PCR; figure 1 with figure 2 ), and a single cell manually sorted from a commercially available cell line (Coriell; 47, XY, +15 or 48, XXY, +21) was used for the first round of whole genome amplification and gene targeting as described in Example 1. Xiang (gene targeting).

[0405] As can be seen, the amplification process results in whole-genome amplification of the genomic DNA and specific amplification of the targeted gene, and amplification of the targeted gene is observed at the selected primer concentration tested. Note that the amplification of WGA in the presence of the gene-specific primer (P) is slightly weaker than the control WGA. These results confirm that overlapping WGA and gene-specific ...

Embodiment 3

[0413] Example 3-Targeting sequence enrichment (TSE) of HBB gene

[0414] In order to further confirm that this procedure can effectively amplify other target sequences, and to evaluate the use of this procedure in genetic screening and / or diagnosis, for example, this procedure is used for preimplantation genetic diagnosis and preimplantation genetic screening. This procedure is used to analyze the enrichment of the target sequence of the HBB gene encoding β globin.

[0415] Multi-cell samples (5-cells) were used to carry out combined whole-genome amplification and HBB gene targeted amplification using two primer sets. Then the PCR products were seeded into two specific HBB PCRs; mainly amplified exons 1 and 2, and amplified exon 3. The second round of HBB PCR amplicons were combined with whole-genome amplified DNA with targeted amplification of the HBB gene to generate a final dilution of 1:20. WGA-only PCR products, combined whole-genome amplification and HBB gene targeted amp...

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Abstract

The present disclosure relates to methods, kits and products for amplifying target DNA sequences from genomic DNA. Certain embodiments of the present disclosure provide a method of amplifying a targetDNA sequence from genomic DNA, the method comprising amplifying the genomic DNA with one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence specific primers, thereby amplifying the target DNA sequence.

Description

[0001] Priority claim [0002] This application claims the priority of Australian Provisional Patent Application No. 2016901197 filed on March 31, 2016, and the content is incorporated herein by reference. Technical field [0003] The present disclosure relates to methods, kits and products for amplifying target DNA sequences from genomic DNA. Background technique [0004] The ability to amplify specific target sequences present in genomic DNA is used in various fields, such as for research purposes, recombinant DNA technology, genetic screening, and for forensic analysis. [0005] Although target-specific amplification is widely used, it has several disadvantages, especially when the amount of starting genomic material is limited. For example, target-specific amplification often results in rapid depletion of limited genomic material, and typically only allows analysis of specific amplified sequences. As a result, it is often difficult to obtain other important genetic information f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q1/686C12Q2525/15C12Q2525/179C12Q2527/107C12Q1/6806C12Q2600/156C12Q2600/16
Inventor 梅林达·贾斯帕米歇尔·弗雷泽
Owner PERKINELMER HEALTH SCIENCES (AUSTRALIA) PTY LTD
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