Whole genome DNA sequence splicing sequencing method

A DNA sequence, whole genome technology, applied in the field of whole genome DNA sequence splicing and sequencing

Inactive Publication Date: 2012-10-17
陈先锋
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  • Claims
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  • Whole genome DNA sequence splicing sequencing method

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Embodiment 1

[0022] 1. Proteinase K / phenol method to extract genomic DNA from blood samples. To ensure the integrity of the DNA (more than 150kb), after phenol extraction, the aqueous phase DNA was transferred to a dialysis bag (dialysis molecular weight cut-off 1000kDa), and dialyzed at 4 degrees Celsius for 3 hours. Subsequent operations will be carried out directly in the dialysis bag until the DNA fragments are attached to the sequencing plate. Before each step of the enzyme reaction, the dialysis bag was dialyzed against 10 times more buffer solution (the same buffer solution as the reaction system) for 30 minutes at 4°C. When performing the enzymatic reaction, place the dialysis bag into a 50ml tube containing a small amount of buffer. After the enzyme reaction, put more than 10 times the amount of buffer in the dialysis bag (matching the next reaction), and perform two dialysis to remove the remaining enzyme and buffer from the previous reaction. For a small amount of DNA samples,...

Embodiment 2

[0043] In the aforementioned operations, the amount of actual DNA fragments used is relatively large, and the preliminary operation process is cumbersome. Therefore, we tried to adopt an improved strategy, adding asymmetric Adapters on both sides of the genomic DNA fragments at the beginning of the experiment. Specific steps are as follows:

[0044] 1. After following step 2 in Example 1, we added Adapter A and A' in a ratio of 1:3, and then added T4 DNA ligase, and the reaction time was 3 hours at 15 degrees Celsius. At this time, the ratio of DNA fragments containing Adapter A at both ends, Adapter A and A' at both ends, and Adapter A' at both ends is 1:6:9 ( figure 2 ).

[0045]2. Import the genomic DNA fragments into the sequencing plate, add T4 DNA ligase, and react at 15 degrees Celsius for 3 hours. At this time, only the DNA fragment containing Adapter A at least at one end is anchored, and the rest of the DNA fragments are still in a free state. After flow wash, o...

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Abstract

The invention which relates to a whole genome DNA sequence splicing sequencing method belongs to the biotechnological field. The invention which improves the preparation of present DNA sequencing substrates, and brings forward a new sequence splicing method can be applied to DNA detection associated with the biology, the medical science, and sidelines of the agriculture, the forestry and the animal husbandry. The invention relates to a series of DNA-associated operations comprising the steps: adding asymmetric junctions to two ends of the genome DNA; anchoring one end to a plane sustenance fully distributed with amplification primers; anchoring the other end of an orientated DNA fragment through methods of electrophoresis and the like; manufacturing and enlarging incisions on a DNA duplex; connecting to 5' terminals of the incisions with sequencing primers containing random ends; connecting the amplification primers with the sequencing primers through a singe-stranded DNA ligase; and increasing the incision number to generate local DNA fragments which can be sequenced. Compared with present sequencing technologies, the method of the invention allows each of the local DNA fragments to reserve distance information, and a complete genome sequence to be obtained through marking splicing results of the local sequences with distances as weights. The method of the invention has protruding advantages in splicing of unknown biological genomes and detection of the genome variation.

Description

[0001] Technical Field: Molecular Biology Technology Background technique [0002] High-throughput whole-genome DNA sequencing has very important applications in biotechnology-related fields. The main purpose of this type of technology is to complete the sequencing of all genomic DNA sequences of a species within a limited time. This type of technology has the characteristics of fast and high sensitivity, and can obtain a wealth of species genome sequence information; therefore, in many fields such as medical related detection, rapid identification of agricultural diseases and insect pests, criminal investigation and forensic identification, etc., this type of technology has great commercial value. Application prospects. In practical application, the existing whole-genome high-throughput sequencing technology can be called "second-generation whole-genome sequencing technology". In the experimental process of "quantification", many small fragment sequences (30 to 100 bases in...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 陈先锋
Owner 陈先锋
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