Compositions, kits, methods and systems for nucleic acid sample amplification

A nucleic acid sample and composition technology, applied in the field of nucleic acid sample amplification composition, can solve the problems of difficulty in implementing multiple PCR capture technology, primer coverage, uniformity, etc.

Pending Publication Date: 2022-07-01

GUANGDONG FAPON BIOTECH CO LTD

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AI-Extracted Technical Summary

Problems solved by technology

However, the more target regions to be captured by multiplex PCR, the coverage and uniformity of primer am...

Method used

3, adopt the composition of embodiment 10, obtain the amplicon sequencing library that constructs, carry out the detection of library size with Agilent2100Bioanalyzer, the result is as shown in Figure 2, library main peak size is at 300-400bp, without obvious small Fragment (primer-dimer or adapter-dimer); quantified by Qubit concentration, the concentration of the constructed amplicon sequencing library is 40-60ng/μL, which shows that the amplification efficiency is high.

The contriver can be used for detecting the primer of target gene variation site through creative design, not only avoids primer dimer, reduces specific amplification, can also be used for multiplex PCR amplification and/or multiple primer composition composition Or amplicon sequencing library construction, unexpectedly high uniformity and/or high coverage, overcoming the difficulties of existing technologies.

[0064] A composition for nucleic acid sample amplification, including primer set 9. Among them, Phenylketonuria (Phenylketonuria) is a disease caused by PAH gene defects. Primers were creatively designed for 212 pathogenic sites on PAH to avoid primer dimers and specific amplification, taking into account high uniformity and High coverage, and optimized to obtain primer set 9 (nucleotide sequence shown in SEQ ID NO: 163-SEQ ID NO: 190).

[0068] The composition for nucleic acid sample amplification, including primer set 9 and primer set 5. Among them, Glycogen Storage Disease II (Glycogen Storage Disease II) is a disease caused by a defect in the GAA gene. Primers were creatively designed for 75 patho...

Abstract

The invention relates to a composition, a kit, a nucleic acid amplification method, a library building method and a system for nucleic acid sample amplification, which can be used for detection of single gene disease related variation sites, and high homogeneity and/or high coverage can be obtained while primer dimer and non-specific amplification are avoided.

Application Domain

Microbiological testing/measurementDNA/RNA fragmentation

Technology Topic

Primer dimerMolecular biology +3

Image

Examples

Experimental program(11)
Effect test(1)

Example Embodiment

[0063] Example 1

[0064] Compositions for amplification of nucleic acid samples, including primer set 9. Among them, phenylketonuria (Phenylketonuria) is a disease caused by PAH gene defect. Primers are creatively designed for 212 pathogenic loci on PAH to avoid primer dimers and specific amplification, taking into account high homogeneity and High coverage, and optimized to obtain primer set 9 (nucleotide sequences shown in SEQ ID NO: 163-SEQ ID NO: 190).

Example Embodiment

[0065] Example 2

[0066] A composition for nucleic acid sample amplification, including primer set 9 and primer set 2. Among them, 21-hydroxylase deficient congenital adrenal hyperplasia (Adrenal hyperplasia, congenital, due to 21-hydroxylasedeficiency) is a disease caused by CYP21A2 gene defect, and primers are creatively designed for 10 pathogenic loci on CYP21A2 , avoiding primer-dimer and specific amplification, taking into account high uniformity and high coverage, and optimized to obtain primer set 2 (nucleotide sequence shown in SEQ ID NO:47-SEQ ID NO:60); primers Group 9 is the same as Example 1.

Example Embodiment

[0067] Example 3

[0068] A composition for nucleic acid sample amplification, comprising primer set 9 and primer set 5. Among them, Glycogen Storage Disease II (Glycogen Storage Disease II) is a disease caused by GAA gene defect. Primers are creatively designed for 75 pathogenic loci on GAA to avoid primer dimerization and specific amplification. Taking into account high homogeneity and high coverage, primer set 5 (nucleotide sequences shown in SEQ ID NO: 97-SEQ ID NO: 138) was obtained by optimization; primer set 9 was the same as Example 1.

PUM

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