Genotypes can be assayed by a variety of experimental methods, but assays for haplotype analysis, such as molecular haplotype analysis, are limited. Dependent on the degree of linkage disequilibrium (L D ) between markers, haplotypes can be inferred from genotype data within certain confidence intervals. We have developed a method for molecular haplotype analysis by linking-emulsion polymerase chain reaction (L E -P C R ) that can be utilized when L D is limited, particularly when the polymorphism under study affects the function of a single gene product. We have used this technique to study the human throw raoxomwe J gene (P O M ) , in which polymorphisms affect transcriptional and enzymatic activity, exhibiting incomplete L D . P O N l are enzymes with a variety of activities, including detoxification activity against organophosphates.
By Martin, this experiment is from "Environmental Genomics Experiment Guide".
Operation method
Experimental molecular haplotype analysis method based on emulsion polymerase chain reaction Move The L E -P C R technique will be interpreted by means of two common genetic polymorphisms, P O M -909 g> c and Q 192R, spaced 15 k b apart on the human] (P O M ) gene. The P O M -909 g > c polymorphism and another functional promoter polymorphism located at one 108 that affects the extent of transcription present an almost complete L D . Q 192R affecting substrate specificity. In a study with a total of 37 877 participants there were mixed heterozygous genotypes at both loci resulting in mixed haplotypes. We determined the molecular haplotypes of these individuals and measured enzyme activities (phenotypes) with both substrates. We also performed molecular haplotype analysis of the third polymorphism, L 55M , using cadaver 〇 ]\0-909§><: and Q 192R for individuals with mixed heterozygous genotypes at both L 55M and the second locus. Only two molecular haplotype analyses were required to determine individual haplotypes that were heterozygous at all three loci. By comparing the predictive power of molecular haplotype analysis with that of putative haplotypes for phenotyping, we demonstrated the utility of L E -P C R population studies (1). This chapter deals only with the L E -P C R for P O M -909 g> c and Q 192R, and does not address phenotypic measurements. The whole idea of L E -P C R is shown in Fig. 1. Two amplicons spanning the polymorphic region are generated in a water-soluble droplet of emulsion as shown below the template. The linkage P C R joins these amplicons to form a microchromosome and retains the information associated with the two polymorphisms. 3.1 Primer design 1. Two primers are required for each amplicon in the emulsion P C R as shown in Figure 2A. Amplicons should be limited to 200 nucleotides. 2. The external primers are typical PCR primers, approximately 25 nt. In our experience, primers with 50% GC and ending in A A are preferred. 1. Capped oligonucleotides are used as shown in Figure 4B. Two products are retained after step 3. 5, including the microchromosome as well as the single-stranded byproduct that escapes from the P C R removal process. Phosphorylation of the 3' end of the capped oligonucleotides prevents them from acting as primers. Instead, they serve as templates to extend the byproducts in case they act as primers for P C R and create new microchromosomes. 2. Our experience suggests that capping of the byproducts is indispensable to prevent the formation of new microchromosomes. When Information about each phase of the polymorphism. So for every two alleles there are two possible pairs of microchromosomes. 2. The calculations assume that all q A S P C R primers are equally efficient in amplifying P C R corresponding templates, as in our four examples. If they are not, appropriate corrections need to be made. 3. Calculate the average C (value for the first pair of haplotypes: here 19277-909c+192 C /-909 g . 4. Calculate the average (^ value for the second pair of haplotypes: here 192T/-909 g +192C/-909c. 5. Subtract to obtain A C z. 6. It is required that the two G values of the first pair of possible haplotypes should be smaller than the two Q values of the second pair of possible haplotypes; or the former should be larger than the latter. Thus one of the four q A S P C R measurements will always have a haplotype that is large. 7. The value of A C i must be greater than 1 or less than 1. Thus q A S P C R has a much greater preference for one pair of haplotype measurements than for the other pair. 8. If conditions 6 and 7 are met, choose the lowest ( ¾ ) value. 9. If conditions 6 and 7 are not met, see Notes 3 to 6. 1. The tube must be in contact with the platform. 2. A device that suspends the tube from the perimeter to dispense the emulsion is not effective. 3. if the observed value of A C t is close to 0 , the L E -P C R fails, the 4. Early in the development of this technique, the failure rate of LEPCR was as high as 20% to 30%, but with improved emulsion formation, the failure rate was reduced to less than 5 %. 5. We found that all these failures occurred at the emulsion PCR step and could not be rescued by the purification and analysis steps. 6. if one haplotype cannot be obtained, redo all L E - P C R experiments again For more product details, please visit Aladdin Scientific website.
3.6 Capping of by-products
greater than the experimental error of the technique.