Quality control standard product for checking 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 false positives in sequencing, loss of copy number information, and uneven amplification.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0033] Embodiment one: the preparation of standard substance
[0034] 1. Design and synthesis of ssDNA nucleic acid sequence
[0035] Design two ssDNA nucleic acid sequences, which are respectively called sequence F and sequence R, and the specific base sequences and various parts of the sequences are as follows.
[0036]
[0037] 2. Dissolving ssDNA Nucleic Acid Sequence
[0038] Sequences F and R were prepared as 10 uM solutions, respectively.
[0039] 3. Formation of hybridization reaction standard
[0040] Mix the dissolved F and R in equal molar concentrations, incubate at room temperature for 2 hours, then preliminarily identify the hybridization status by 2% agarose gel electrophoresis, and recover the hybridized strands to obtain the standard.
Embodiment 2
[0041] Example 2: Verification after standard preparation
[0042] 1. Standard verification step 1
[0043] The above-mentioned prepared products were identified by filling gaps with two DNA polymerases with different reaction properties, DNA polymerase I and Q5 DNA polymerase, respectively.
[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' end and 3' end is obtained, which is called D1, Schematic such as image 3 As shown; Q5 DNA polymerase fills up the gap through its polymerase activity and at the same time performs a strand displacement reaction to obtain a dsDNA sequence that changes from a dumbbell-shaped structure to a full-length linear length, which is called Q5. The schematic diagram is as follows Figure 4 shown. The sequence obtained after the action of the corresponding enzyme was verified by Sanger sequencing. ...
Embodiment 3
[0047] Embodiment three: application after preparation of standard substance
[0048] 1. In vitro transcription and amplification of fragmented nucleic acids
[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 the transcription promoter sequence B and initiate transcription downstream of it, such as Figure 7 As shown, a single-stranded RNA sequence corresponding to the downstream single-stranded sequence of known sequence B is 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 types of products such as Figure 8 show...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More