A Method for Detecting Discontinuous Multiple DNA Sites by Combining Overlap Extension PCR with Sanger Sequencing
An overlapping extension and purpose technology, which is applied in the field of overlapping extension PCR combined with Sanger sequencing to detect discontinuous multiple DNA sites, and can solve the problem that the compound directional extension multiple DNA fragment PCR technology has not yet appeared.
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Embodiment 1
[0049] The design and PCR reaction of the amplification primer of embodiment 1 full-length
[0050] 1. Primer design
[0051] According to the primer design principles and the primer design requirements of overlap extension PCR, 2 universal universal full-length amplification primers were designed: the universal universal full-length amplification primer is a 22bp DNA that is not complementary to human genes, designed by ourselves , and screened according to amplification efficiency and amplification specificity to obtain the most suitable primer, the nucleotide sequence of which is shown in SEQ ID No.1-2,
[0052] SEQ ID No.1: 5'-CTGCATAGTCTACTGGATCAAC-3';
[0053] SEQ ID No.2: 5'-GATCGTCAGTGCTCACTACATC-3';
[0054] Design of fragment amplification primers (overlapping target DNA fragments) for amplifying each target fragment (taking the connection of two target DNA fragments as an example):
[0055] The 5' end upstream fragment amplification primer of the most upstream am...
Embodiment 2
[0066] Embodiment 2 A kind of method for detecting multiple DNA fragment sites
[0067] 1. Amplification of multiple DNA fragments
[0068] The PCR amplification of the DNA fragment site was performed according to the multiple DNA fragment site overlap extension PCR reaction in Example 1.
[0069] 2. Detection
[0070] The amplified product of Example 1 was subjected to Sanger sequencing using primers with nucleotide sequences such as those shown in SEQ ID No. 1-2.
[0071] Preferably, the reagent for Sanger sequencing is BigDye Terminator v3.1CycleSequencingKit.
[0072] More preferably, the PCR reaction system for Sanger sequencing is: 0.5 μl of BigDye; 1.75 μl of 5×seq Buffer; 1 μl of each primer; 1 μl of template; and 10 μl of water.
[0073] Preferably, the program of the PCR reaction for Sanger sequencing is: 96°C for 1min; 96°C for 10s, 50°C for 5s, 60°C for 4min, 25 cycles; 4°C∞.
Embodiment 3
[0074] Embodiment 3 A kind of kit of PCR amplification of multiple DNA fragments
[0075] A kit for PCR amplification of multiple DNA fragments,
[0076] Including universal full-length amplification primers whose nucleotide sequences are shown in SEQ ID No.1-2 and fragment amplification primers for amplifying each target fragment;
[0077] The 5' end upstream fragment amplification primer of the most upstream amplified fragment is the 5' end of the conventionally designed upstream primer of the fragment plus the nucleotide sequence as shown in SEQ ID No.1,
[0078] The upstream fragment amplification primer at the 5' end of the remaining amplified fragments is the 5' end of the conventionally designed upstream primer of the fragment plus 18 to 23 bases at the 3' end of the upstream target fragment,
[0079] The downstream fragment amplification primer at the 3' end of the most upstream amplified fragment is a conventionally designed downstream primer 5' end plus the nucleoti...
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