Multiple PCR primer group for human HNF-1 alpha gene amplification

A technology of HNF-1 and α gene, applied in the field of primer library for amplifying human HNF-1α gene

Inactive Publication Date: 2004-02-11
SAMSUNG ELECTRONICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Multiplex PCR also costs more cost, material resources and time

Method used

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  • Multiple PCR primer group for human HNF-1 alpha gene amplification
  • Multiple PCR primer group for human HNF-1 alpha gene amplification
  • Multiple PCR primer group for human HNF-1 alpha gene amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1: Preparation of primers for amplifying 10 target sequences in the human HNF-1α gene

[0073] Primers were designed so that each final PCR product had at least a 5-10 bp difference in size. The aforementioned factors need to be considered when designing a set of primers for multiplex PCR. Further, each primer should be designed not to include 4 or more identical consecutive nucleotides.

[0074] Primer analysis using HYBsimulaor TM (Advanced Gene Computing Technologies, Inc).

[0075] In order to improve the amplification efficiency of PCR products, T7 promoter sequence (SEQ ID NO.1) was added to the 5' end of the forward primer, and T3 promoter sequence (SEQ ID NO.2) was added to the 5' end of the reverse primer .

[0076] The sequence numbers and properties of each primer are given in Table 1.

[0077] Primer

[0078] F: Forward R: Reverse

Embodiment 2

[0079] Example 2: Amplification of the Human HNF-1α Gene Using Single PCR

[0080] Each target sequence of the human HNF-1α gene was amplified by a single PCR using each set of primers of Example 1. PCR is completed under the following conditions, pre-denaturation (95°C for 5 minutes), 30 cycles of denaturation (95°C for 30 seconds), annealing (64°C for 15 seconds), polymerization (72°C for 30 seconds), and finally extension (72°C for 3 seconds). minutes) PCR reaction composition is as follows:

[0081] Sterile DNase-RNase-free water 12.8 μl

[0082] dNTP mixture (each nucleotide 2.5mM) 2μl

[0083] 10×Taq polymerase buffer 2μl

[0084] A set of primers (each 10pmol) 2μl

[0085] Genomic DNA (200-1.0μg) 1μl

[0086] Taq polymerase (5 units / μl) 0.2μl

Embodiment 3

[0089] Example 3: Amplification of Human HNF-1α Gene by Multiplex PCR

[0090] Multiplex PCR was performed using a set of primers from Example 1. PCR was completed under the following conditions, pre-denaturation (95° C. for 5 minutes). 30 cycles, denaturation (95°C for 30 seconds), annealing (64°C for 15 seconds), polymerization (72°C for 30 seconds), and final extension (72°C for 3 minutes), all primers were added in one reaction tube, and the reaction set The points are as follows:

[0091] Sterile DNase- and RNase-free water 18.4 μl

[0092] dNTP mixture (2.5mM each nucleotide) 5μl

[0093] 10×Taq polymerase buffer 5μl

[0094] Primers (each 10pmol) 20μl

[0095] Genomic DNA (200-1.0μg) 1μl

[0096] Taq polymerase (5 units / μl) 0.6μl

[0097] The PCR products were analyzed by electrophoresis on 1.8% agarose gel (Figure 1, lane 12). In Fig. 1, using the primer set of Example 1 to perform multiplex PCR results, all 10 target sequences (579, 459, 365, 308, 332, 241, 28...

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Abstract

The present invention provides a primer pool for amplifying a target sequence of a human HNF-1 alpha gene including at least one set of primers or variant primers thereof, each set of primers being identified by two consecutive SEQ ID NOS, the SEQ ID NOS starting at SEQ ID NO.3 and terminating at SEQ ID NO.22 according to the present invention. A target sequence such as the human HNF-1 alpha gene may be amplified with a high specificity, a high speed, a high sensitivity and a low cost through a multiplex PCR to detect a maturity-onset of diabetes of the young (MODY) 3 associated gene.

Description

field of invention [0001] The present invention relates to a primer pool for amplifying human HNF-1α gene, more specifically, a primer pool for amplifying target DNA sequence in human HNF-1α gene by multiplex PCR (multiplex PCR). Background technique [0002] One class of methods for detection of hybridizing nucleic acids includes the polymerase chain reaction (PCR). PCR methods are well known in the art (U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159). In PCR, the nucleic acid primers are complementary to the opposite strands of the amplified target sequence, and the nucleic acid primers anneal to the denatured sample. A DNA polymerase (typically thermostable) then extends the double strand of DNA from the hybridized primer. This process is repeated continuously to amplify the target nucleic acid. If the nucleic acid primer cannot hybridize to the sample, no corresponding PCR amplification product can be formed. In this case, the PCR primers serve as hybridization pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53C12N15/09C12Q1/68G01N33/566
CPCC12Q1/6883C12Q2600/16C12Q1/6827C12Q1/6853C12Q2527/101C12Q2527/113C12Q2537/143C12Q1/68
Inventor 李娟受金美卿李贞男
Owner SAMSUNG ELECTRONICS CO LTD
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