Multiplex pcr primer set for human glucokinase gene amplification
A glucokinase and gene technology, which is applied in the field of primer sets for glucokinase genes, can solve the problems of time-consuming and energy-consuming and high cost of multiplex PCR
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Embodiment 1
[0049] Example 1: Preparation of primers for the amplification of 11 target sequences of the human glucokinase gene
[0050] Primers were designed such that the size of each PCR product differed by at least 5-10 bp. Consider the above factors when designing primer sets for multiplex PCR. In addition, each primer was designed not to include consecutive 4 or more identical nucleotides.
[0051] Use HYBsimulator TM (Advanced Gene Computing Technologies, Inc) to analyze the primers.
[0052] In order to improve the amplification efficiency of the PCR product, a T7 promoter sequence (sequence 1 (SEQ ID NO.1)) was added at the 5' end of the forward primer, and a T3 promoter sequence (sequence 2 (SEQ ID NO.1)) was added at the 5' end of the reverse primer. SEQ ID NO. 2)).
[0053] The sequence numbers and characteristics of each primer are shown in Table 1.
[0054] Primer
[0055] F: Forward R: Reverse
Embodiment 2
[0056] Example 2: Amplification of the human glucokinase gene by single-pass PCR
[0057] Using each set of primers of Example 1, each target sequence of the human glucokinase gene was amplified by a single PCR. PCR is implemented as follows: first denaturation (95°C for 5 minutes), then denaturation (95°C for 30 seconds), annealing (64°C for 15 seconds) and polymerization (72°C for 30 seconds) as a cycle, a total of 30 cycles, and finally extension (72°C for 3 minutes). The composition of PCR reaction solution is as follows:
[0058] Sterile DNase- and RNase-free water 12.8 μl
[0059] dNTP mix (2.5mM for each nucleotide) 2μl
[0060] 10x Taq polymerase buffer 2μl
[0061] A set of primers (each primer 10pmol) 2μl
[0062] Genomic DNA (200-1.0μg) 1μl
[0063] Taq polymerase (5 units / μl) 0.2μl
[0064] PCR products were analyzed by electrophoresis on a 1.8% agarose gel (Fig. 1). In Figure 1, lanes 1-11 represent PCR products corresponding to exons 1a, 1b, 1c, 2, 3, 4...
Embodiment 3
[0068] Example 3: Amplification of the human glucokinase gene by multiplex PCR
[0069] Multiplex PCR was performed using a set of primers from Example 1. The reaction was carried out as follows: first denaturation (5 minutes at 95°C), followed by 30 cycles of denaturation (30 seconds at 95°C), annealing (15 seconds at 64°C) and polymerization (30 seconds at 72°C). Extension (3 min at 72°C). All primers are added to one reaction tube. The composition of this reaction solution is as follows:
[0070] Sterile DNase- and RNase-free water 16.4 μl
[0071] dNTP mix (2.5mM for each nucleotide) 5μl
[0072] 10x Taq polymerase buffer 5μl
[0073] A set of primers (each primer 10pmol) 22μl
[0074] Genomic DNA (200-1.0μg) 1μl
[0075] Taq polymerase (5 units / μl) 0.6μl
[0076] PCR primers were analyzed by electrophoresis on a 1.8% agarose gel (Figure 1, lane 11). As shown in Figure 1, the result of multiplex PCR using the above-mentioned set of primers of Example 1 is that a...
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