Analysis of predisposition based on buman airway tripsin protease gene polymorphism
A technology of gene polymorphism and trypsin, which is applied in the field of physical fitness analysis based on the polymorphism of the human respiratory tract trypsin gene, can solve the unexplained human respiratory tract trypsin-like enzyme and other problems
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[0041] The present invention will be described in detail through examples below, provided that these examples are not intended to limit the method of the present invention. The method is to predict disease-related constitution by analyzing the gene polymorphisms of human airway trypsin-like enzymes.
[0042] 〔Standard Operating Method〕
[0043] Standard DNA extraction procedures, gene amplification procedures, restriction enzyme digestion procedures and electrophoresis procedures that can be used in the present invention will be described below.
[0044] (a) Standard DNA extraction operation
[0045] There is no particular limitation on the biological sample used for the gene amplification of the present invention, however, blood cell components are suitable because this sample is easily obtained and its DNA is easy to extract.
[0046] 1. 0.5 ml of whole blood (anticoagulated with 2Na-EDTA) was placed in a microcentrifuge tube with a capacity of 1.5 ml.
[0047] 2. Add 0....
example
[0049] Solvent example: 1×SSC
[0050] Preparation of this solvent: 2OxSSC adjusted to pH 7.0 by 10-fold dilution with 10N NaOH containing 175.3g NaCl and 88.2g sodium citrate in 1 liter.
[0051] 3. The mixture was centrifuged (10,000 g for 20 seconds at 4°C) and the supernatant was carefully removed without removing the dark pellet.
[0052] 4. 1 ml of solvent was added and the mixture was stirred.
[0053] 5. The mixture was centrifuged (10000 g, 20 seconds at 4°C), and the supernatant was removed.
[0054] 6. Repeat steps 4 and 5 one more time.
[0055] 7. Add 200 μl of enzyme reaction solution and 10 μl of proteolytic enzyme and mix immediately.
[0056] An example of an enzyme reaction solution: a mixture of 0.04M DTT (dithiothreitol) with 0.2M NaOAc (sodium acetate) and 0.4% SDS.
[0057] Examples of proteolytic enzyme solutions:
[0058] 10mg / ml protease (Protease K)
[0059] 8. The mixture was incubated at 37°C for 1 hour (the mixture was mixed by gentle s...
Embodiment 1
[0082] [Example 1] Obtaining a DNA fragment containing an intron on the human airway trypsin-like enzyme genome and analyzing its base sequence
[0083] In order to search for the intron site on the genome of human airway mature trypsin-like enzymes, several primers were prepared to amplify human genomic DNA with reference to the composition of exons and introns in the genomes of several trypsin-like enzymes. A DNA fragment (gene fragment) of about 6Kb was amplified by primers A1 (SEQ ID NO: 7) and A2 (SEQ ID NO: 8), and a DNA fragment (gene fragment) of about 1.5 Kb was amplified by primers B1 (SEQ ID NO: 9) and B2 (SEQ ID NO: 10). A DNA fragment of Kb, a DNA fragment of about 3.4Kb was amplified by primers C1 (SEQ ID NO: 11) and C2 (SEQ ID NO: 12). ( figure 1 )
[0084] DNA fragments were excised from the gel and purified, and each fragment was inserted into a TA cloning vector (manufactured by Invitrogen). The nucleotide sequences on both sides (5'-end and 3'-end) of the...
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