A set of primer combinations and kits for simultaneous detection of 93 bovine genetic defect genes and lethal haplotypes
A genetic defect and kit technology is applied in the field of a set of primer combinations and kits for simultaneous detection of 93 bovine genetic defect genes and lethal haplotypes, which can solve the problems of easy introduction of errors, misjudgments, poor repeatability, etc. Detecting difficult effects
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Embodiment 1
[0082] Example 1: Construction of a method for simultaneous detection of 93 bovine genetic defect genes
[0083] 1. PCR amplification reaction primer design and condition optimization
[0084] Organize the SNP sequence information into a standard format, design PCR amplification reaction primers and single-base extension primers for 93 causal mutation sites (SNPs, insertions or deletions of short fragments), and check the conditions that affect the detection reaction system and PCR procedures. Optimization, including reagent concentration, temperature, time, number of reaction cycles, etc., and then a 384-well PCR reaction (93 causal mutation sites were divided into 4 PCR wells for simultaneous detection). The PCR amplification reaction is as follows:
[0085] (1) Use AssayDesigner3.1 software to design primers for each mutation site sequence, analyze the primers after the optimization test, and mix the PCR primers required for detecting mutation sites in each well with deion...
Embodiment 2
[0104] Embodiment 2: The kit for detecting 93 kinds of bovine genetic defect genes
[0105] The composition of the kit includes: 1-4 well amplification primer mix (500nM each), 1-4 well extension primer mix (7μM: 14μM), dNTP (25mM each), MgCl 2 (25mM), 0.100μl HotStar Taq (5U / μl), PCR Bμfferwith 15mM MgCl 2 , Shrimp Alkaline Phosphatase Buffer, Shrimp Alkaline Phosphatase (1U / μl), iPLEX Bμffer Plus, iPLEX Termination mix, iPLEX Enzyme, ddH 2 O, 384Dimple board, 384 sample board, resin.
[0106] Wherein, the sequence of the amplification primer mixture and the extension primer mixture described in the kit is as described in the summary of the invention above. The detection steps of bovine tissue samples using the kit are as follows:
[0107] (1) Use the reagents in the kit to carry out PCR amplification reaction on the DNA in bovine blood, semen or hair follicles qualified for quality control. The causal mutation sites of 93 kinds of bovine genetic defects in each sample are...
Embodiment 3
[0116] Example 3: Sample detection of different genetic defect genes and characteristic milk genes of cattle
[0117] The blood samples of 96 Holstein cows were collected, and the causal mutation sites (SNP, insertion or deletion of short fragments) of the cow samples were detected by using the reagents in the kit of Example 2 of the present invention and related steps.
[0118] It was found that some cattle carried (heterozygous) genetic defect genes and lethal haplotypes ( Figure 3-Figure 10 ) and verified by sequencing. The genotype detection results of the specific samples are shown in Table 5 below:
[0119] Table 5. Detection results of bovine genetic defect genes and lethal haplotypes (phenotype traits)
[0120] Genetic defect (phenotypic trait) Number of samples (genotype) Lethal Haplotype (HH1) 3 heads carry Lethal Haplotype (HH3) 7 heads carry Spinal deformity syndrome (HHC) 4 heads carry Recessive Red Hair 373 2 carry ...
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