PCR amplification system and library construction method for whole genome bisulfate sequencing
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A bisulfite, whole genome technology, applied in the field of molecular biology, can solve the problems of unreachable detection concentration, few PCR products, low DNA concentration, etc., to reduce workload, improve work efficiency, and reduce consumption. Effect
Inactive Publication Date: 2016-06-08
SHANGHAI PASSION BIOTECHNOLOGY CO LTD
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However, according to the current conventional method, most of the DNA has been destroyed after salt treatment, and the concentration of the purified DNA is already very low. minimum requirements
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Embodiment 1
[0034] Reagents and kits used in this embodiment:
[0035] (1) Agilent: high-fidelity CX hot start DNA polymerase (PfuTurbocxHotstartDNAPolymerase)
[0037] (3) IllumineDNA Library Construction Kit (TruSeqDNASamplePreparationLowThroughput(LT)Kit)
[0038] 1. Quantification of DNA input amount
[0039] Note: The starting amount of DNA used in this experiment is 5 μg.
[0040] 2. DNA Fragmentation
[0041] (1) Turn on the ultrasonic interrupter in advance and cool down to below 6°C;
[0042] (2) DNA sample (tiger frog virus (TFV) DNA) was diluted to 130 μl with 1×TE, mixed with a gun, transferred all the solution to a clean and dry atomizing cup, and interrupted to 200 bp.
[0043] 3. Purification of DNA Fragments
[0044] (1) Add 3 times the volume of PCR-A solution to the fragmented DNA, mix well and transfer to a purification column up to 700 μl each time, centrifuge at 10,000 rpm for 1 ...
Embodiment 2 comparative test example
[0160] A methylation library for whole-genome bisulfite sequencing was prepared by a conventional method, and the rest of the steps were the same as in Example 1, except that in operation step 10, that is, during PCR amplification, the following conventional PCR amplification system was used:
[0161] Aliquot 12 μl of salt-treated and purified DNA into 3 PCR tubes, and the reaction system in each tube is:
[0162] DNA4μl
[0163] UltraPureWater33.75μl
[0164] PfuTurboCxReactionBuffer5μl
[0165] 10mMdNTPMix 1.25μl
[0166] PCR Primer Cocktail 5 μl
[0167] PfuTurboCxHotstartDNAPolymerase 1 μl
[0168] Total Volume50μl
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Abstract
The present invention relates to the technical field of molecular biology, and discloses a PCR amplification system for whole genome bisulfate sequencing, and a library construction method thereof. The PCR amplification system comprises: 19 [mu]l of ultra-pure water; 15 [mu]l of a DNA template; 5 [mu]l of a reaction buffer liquid, 2.5 [mu]l of an upstream primer, 2.5 [mu]l of a downstream primer; 5 [mu]l of 2.5 mM dNTP; and 1 [mu]l of hot-start DNA polymerase. According to the present invention, with the application of the PCR amplification system in the PCR amplification step of the target fragment treated by the salt during the whole genome bisulfate sequencing process, the product concentration of the PCR amplification reaction can be improved, and the sufficient loading amount of the sequencing can be provided; the present invention further provides a bisulfate sequencing library construction method containing the PCR amplification step, wherein the sufficient loading amount of the methylation sequencing library can be constructed and the smooth operating of the whole genome bisulfate sequencing can be easily achieved through the method.
Description
technical field [0001] The invention relates to the technical field of molecular biology, in particular to a PCR amplification system and a library construction method applied in the whole genome bisulfite sequencing technology. Background technique [0002] DNA methylation modification is an important means of gene expression regulation, which plays a vital role in the normal development of individuals. If there is a problem with the methylation regulation mechanism, it can cause abnormal body functions and lead to the occurrence of some diseases. Therefore, The detection of DNA methylation is of great significance to the research of some gene-related diseases. DNA methylation mainly occurs in CpG islands with high GC content. There are many methods of methylation sequencing, such as genome-wide methylation sequencing, methylated DNA co-immunoprecipitation sequencing, methyl-binding protein sequencing, simplified methylation sequencing, etc. [0003] Bisulfite sequencing ...
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