Unphased determine whether a genetic variant is associated with a disease or trait. If association is present, a particular allele, genotype or haplotype of a polymorphism or polymorphism(s) will be seen more often than expected by chance in an individual carrying the trait. case (Schizophrenia) and control (normal). frequency of alleles or genotypes is compared between the cases and controls. One problem with the case-control design is that genotype and haplotype frequencies vary between ethnic or geographic populations.
After the gel electrophoresis, the PCR products were added with 40Î¼l of 100% ethanol and put into -20â„ƒ refrigerator, for at least 2 hours, this is to precipitate out the amplified fragment of DNA. Then, the mixture was centrifuged at 3000rpm for 30 minutes. After removal of the liquid waste, 24Î¼l of 70% ethanol was added to wash the sample, and centrifuged at 3500rpm for 20 minutees. The washing process was repeated once more. After that, the samples were dried under 10Pa of pressure for 20 minutes. 10Î¼l of Milli Q water was added to each of the dried sample and heated at 60â„ƒ for 1 minute for mixing the water and PCR product.
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The material used in the DNA sequencing reactions include: primer, either forward or reverse; 5X Seuqencing buffer (Applied Biosystems); PCR product; and Milli Q water that to make the total volume into 15Î¼l.
The DNA sequencing initiated at 95â„ƒ for 5 minutes. Each cycle contains 30 seconds at 95â„ƒ for denaturation, 30s at annealing temperature for each pairs of primer for annealing of primer to template DNA, 1 minute at 72â„ƒ for elongation of the product. In the experiment, 30 cycles were completed for each pairs of the primers. Following the reaction cycles, there was 5 minutes at 72â„ƒ as final elongation time. And the reaction products were temporarily stored in the machine at 4â„ƒ as the final hold temperature.
0.72Î¼l of primer, 3Î¼l of sequencing buffer, 0.75Î¼l of Big Dye Terminator version 3.1 (Applied Biosystems), 1.5Î¼l of PCR product sample and 9.03Î¼l of Milli Q water were made up into reaction mixture.
Table 2: Primers used in sequencing.
Figure 2: Schematic drawing of DNA sequencing.
Sequencing Product Purification
The sequencing products were added with 48Î¼l of 100% ethanol and put into -20â„ƒ refrigerator, for at least 30 minutes, this is to precipitate out the DNA fragment. Then, the mixture was centrifuged at 3000rpm for 30 minutes. After removal of the liquid waste, 24Î¼l of 70% ethanol was added to wash the sample, and centrifuged at 3500rpm for 20 minutes. The washing process was repeated twice more. After that, the samples were dried under 10Pa of pressure for 20 minutes. 10Î¼l of Hi-Deionized formamide was added to each of the dried sample and heated at 95â„ƒ for 3 minute to denature.
Results generated by Genepop ,
By using Hardy-Weinberg equilibrium, we try to test if the population (Beijing) used in the experiment is in equilibrium. Control sample from normal persons was used to run the Genepop program.
Table 3: Results generated from Genepop. Two estimates of Fis: Weir & Cockerham's (1984) estimate (W&C), and Robertson & Hill's (1984) estimate (R&H). All (Fisher's method): Chi2 : 1.1210; Df : 18.0000; Prob : 1.
Under the principle of probability, if the p value is less than 5 percent, the two numbers are said to be significantly different, the null hypothesis, in this case the random union of gametes, is rejected. From the above table we can see that all SNPs tested have p-value larger than 0.05; that is the population at the selected loci is expected to be in equilibrium. As we will compare the differences of SNPs between Schizophrenia patients and normal persons, the results generated by Genepop can exclude the variation between ethnic or geographic populations.
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The standard error of this estimate is much less that 0.01, and the results from the estimation are expected to be reliable.
Results generated by Haploview 
Linkage disequilibrium describes a situation in which some combinations of alleles or genetic markers occur more or less frequently in a population than would be expected from a random formation of haplotypes from alleles based on their frequencies.
Fig3: Haploview display of Schizophrenia patients
Fig 4: Haploview display of control group