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Method for detecting beta-casein genotype on basis of restriction enzyme digestion

A detection method, casein technology, applied in the direction of biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of unguaranteed and inability to detect different genotypes of β-casein

Inactive Publication Date: 2016-01-06
SHANGHAI PASSION BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current detection method cannot detect different genotypes of β-casein, and cannot guarantee that the milk people drink is A2 milk

Method used

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  • Method for detecting beta-casein genotype on basis of restriction enzyme digestion

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Use primers to carry out PCR amplification of the dairy cow genomic DNA to be detected: take 1ul of DNA template (45ng / ul), add 0.5ul of 0.1U / ul hot-start TaqDNA polymerase, 2.5uM of dNTP1ul, 0.5 of each of 10uM upstream and downstream primers ul, 2×PCRBuffer 8ul, add double distilled water to 20ul to obtain a PCR amplification system, heat the amplification system to 95°C for 5 minutes, then denature at 95°C for 15s, anneal at 52°C for 35s, extend at 68°C for 35s, and self-denature One cycle was counted from the beginning to the end of the extension, and after 30 cycles, the extension was performed at 68° C. for 10 min.

[0050] Use the restriction endonuclease TaqI to digest the PCR amplification product: Take 5ul of the PCR amplification product, add restriction endonuclease TaqI0.05ul, buffer 0.5ul, incubate at 60°C for 4h, heat inactivate at 75°C 30min.

[0051] Test results such as figure 1 As shown, the amplified product will produce a 218bp fragment and a 35bp...

Embodiment 2

[0053] Utilize primers to carry out PCR amplification of the dairy cow genomic DNA to be detected, take DNA template 1ul (50ng / ul), add 0.1U / ul hot-start TaqDNA polymerase 1ul, 2.5uM dNTP1ul, 10uM upstream and downstream primers each 1ul, 2×PCRBuffer10ul, add double-distilled water to 20ul to obtain a PCR amplification system, heat the amplification system to 95°C for 5 minutes, then denature at 95°C for 15s, anneal at 61°C for 30s, and extend at 72°C for 30s. The end of the extension was counted as one cycle, and after 35 cycles, it was extended at 72° C. for 7 min.

[0054] Use restriction endonuclease TaqI to carry out enzyme digestion, judge according to the result of agarose gel electrophoresis, take 9ul of PCR amplification product, add restriction endonuclease TaqI0.2ul, buffer solution 1ul, incubate at 65 ℃ for 3h, in Heat inactivation at 80°C for 20min.

[0055] Test results such as figure 1 As shown, the amplified product will produce a 218bp fragment and a 35bp sm...

Embodiment 3

[0057] Use primers to perform PCR amplification of the cow genomic DNA to be detected, take 1ul of DNA template (55ng / ul), add 0.1U / ul hot start TaqDNA polymerase 1.5ul, 3uM dNTP1.5ul, 15uM / ul upstream and downstream 1.5ul of each primer, 12ul of 2×PCRBuffer, add double distilled water to 20ul to obtain a PCR amplification system, heat the amplification system to 95°C for 5min, then denature at 92-95°C for 15s, anneal at 65°C for 25s, and 72°C Extend for 25 s, and count as one cycle from the beginning of denaturation to the end of extension, and after 45 cycles, extend at 72°C for 4 min.

[0058] Use restriction endonuclease TaqI to carry out enzyme digestion, judge according to the result of agarose gel electrophoresis, take PCR amplification product 13ul, add restriction endonuclease TaqI0.35ul, buffer 1.5ul, incubate at 65 ℃ for 3h, Heat inactivation at 80°C for 20min.

[0059] Test results such as figure 1 As shown, the DNA amplification product containing A1β-casein cow...

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Abstract

The invention discloses a method for detecting beta-casein genotype on the basis of restriction enzyme digestion. The method comprises the following steps of designing a primer by which amplified fragments of an A1 type beta-casein gene contain TaqI cleavage sites but amplified fragments of an A2 beta-casein gene do not generate TaqI cleavage sites according to a base sequence of the beta-casein gene; performing PCR (polymerase chain reaction) amplification on a to-be-detected dairy cow genome DNA (deoxyribonucleic acid) by using the primer; performing digestion on amplified products by using restriction enzyme TaqI; and judging according to a result of agarose gel electrophoresis. The amplified products which contain A1 beta-casein can generate a 218 bp fragment and a 35 bp small fragment after digestion; and the amplified products of the A2 beta-casein gene cannot generate the TaqI digestion sites, cannot be cut off by TaqI, and are still 253 bp bands. By the method for detecting the beta-casein genotype on the basis of restriction enzyme digestion, different genotypes of the beta-casein can be detected, so that milk consumed by people contains A1 type beta-casein.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting β-casein genotype based on enzyme digestion. Background technique [0002] There are two main types of protein in milk: casein and whey. Casein accounts for about 80% of milk protein, and whey protein accounts for about 14%. Milk protein generally contains four kinds of casein, αS1-casein, αS2-casein, β-casein and κ-casein, of which β-casein accounts for about 25-35% of the total casein. [0003] β-casein contains 209 amino acids, and it contains at least 13 different amino acid compositions, so it is divided into A1, A2, A3, B, C, D, E, F, H1, H2, I, G. At present, the β-casein produced by most farmed dairy cows is mainly A1 and A2. The main difference between A1 and A2 type β-casein is the difference in the 67th amino acid, the 67th position of A1 β-casein is histidine (codon is CAT), and the same position of A2 β-casein is proline ( codon is CCT). [00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/44
Inventor 孙子奎高文学丁方美王锋方钰
Owner SHANGHAI PASSION BIOTECHNOLOGY CO LTD
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