Quality control standard for testing nucleic acid amplification uniformity and preparation method thereof
A standard and quality technology, applied in the field of genetic engineering, which can solve the problems of loss of copy number information, uneven amplification, and difficulty in error correction.
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Embodiment 1
[0033] Example 1: Preparation of standard product
[0034] 1. Design and synthesize ssDNA nucleic acid sequences
[0035] Two ssDNA nucleic acid sequences were designed, respectively called sequence F and sequence R. The specific base sequences and various parts of the sequences are as follows.
[0036]
[0037] 2. Solubilize ssDNA Nucleic Acid Sequences
[0038] Sequences F and R were prepared as 10 uM solutions, respectively.
[0039] 3. Hybridization Reaction Standard Formation
[0040] Mix the dissolved F and R in equimolar concentrations evenly, incubate at room temperature for 2 hours, and then preliminarily identify the hybridization situation by 2% agarose gel electrophoresis and gel recovery of the hybridized strand to obtain the standard product.
Embodiment 2
[0041] Example 2: Verification after standard preparation
[0042] 1. Standard product verification step 1
[0043] The above-prepared products are respectively identified by filling gaps with two DNA polymerases with different reaction properties, DNA polymerase I and Q5 DNA polymerase.
[0044] like figure 2 As shown, DNA polymerase I fills the gap through its polymerase activity, and through its gap translation activity, after its action, a DNA fragment without Tran / Tran* at the 5' and 3' ends is obtained, which is called D1, Schematic such as image 3 Q5 DNA polymerase, while filling the gap through its polymerase activity, carries out strand displacement reaction to obtain a dsDNA sequence with a full-length linear length from a dumbbell-shaped structure, which is called Q5. The schematic diagram is as follows. Figure 4 shown. The sequence obtained by the corresponding enzyme was verified by Sanger sequencing.
[0045] 2. Standard product verification step 2
[0046...
Embodiment 3
[0047] Example 3: Application after standard preparation
[0048] 1. Fragmented nucleic acid in vitro transcription amplification
[0049] Transcription amplification is to pre-amplify the fragmented nucleic acid so that its quantity meets the requirements for sequencing library construction. In vitro transcription can convert a double-stranded DNA into multiple single-stranded RNAs to achieve the effect of nucleic acid amplification. When T7 RNA polymerase is used for in vitro transcription, it can bind to transcriptional promoter sequence B and initiate transcription downstream of it as Figure 7 As shown, a single-stranded RNA sequence corresponding to the single-stranded sequence downstream of the known sequence B was formed. Since there are two B sequences on the fragmented DNA, T7 RNA polymerase can have two different transcription directions, and in vitro transcription will generate two different products such as Figure 8 shown. In vitro transcription is an amplifi...
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