Week 10 Attachment Sharing

Hi guys!! It is my turn to blog again! For the past few weeks I have been handling 2 projects on allele discrimination. As mentioned previously in my blog, I used allelic discrimination kit to identify the different alleles present in the samples provided. However, there is another method to differential the allele, namely the Enzyme-based method. This method involved a lot of molecular biology principle and is quite time consuming too.


Despite that the duration for this method is rather long, the results produced is much more easier to analyze and interpret. Nevertheless, this method is not suitable for most of allele discrimination. It can only be used if the polymorphism of the allele can be ‘coincidently’ digested by any specific restriction enzymes. In my case, it happens that the alleles we emphasized are different by a nucleotide and can be digested by a particular enzyme.



Recipe for Enzyme-based methods

1.First and foremost, the DNA samples are amplified by using the PCR thermocycler and specific primers (both forward and reverse) close to the target gene.
2.The DNA samples then undergoes restriction digestion performed by a specific restriction enzyme (it is very important that the restriction enzymes cutting site matches with the polymorphism).
3. Digested samples are run in gel for separation of alleles
4.Stained the gels in EtBr (Recommendation: add EtBr when casting gels, it is a lot better!!)
5. The gels are then detected under UV light.



Interpretation of results:

Since the restriction enzymes is able to use the polymorphism as it cutting site, only DNA samples that comprised of the allele will be digested. For example if allele y contains the restriction enzymes cutting site (contains the target nucleotide), restriction enzymes will be able to cut the allele into two fragments. In contrast, if it is allele Y then there will be no cutting site for the enzymes to act on. As a result, allele y will appear below allele Y after electrophoresis. This is because allele y has been digested into smaller fragments and hence will drift faster than allele Y in the gels. Then again if the DNA samples is a heterozygous, there will be a double bands. Since only one of the chromosomes contains the cutting site, this amount of chromosomes digested will also be half while the rest remain uncut. Hence, a double band will appear as half of the digested fragment (digested one) drift faster than another half (undigested).



Other methods for allele discrimination:

Dynamic allele-specific hybridization (DASH)
Basically this method worked by measuring the difference in melting temperate in the DNA samples. Unlike the other methods, DASH used biotinylated primer with a bead attached to it. The biotinylated primer with the bead is then extend the DNA samples is amplified.
The amplified DNA samples is then incubate in streptavidin column. NaOH is then added to wash away the unbiotinylated DNA. This is because the unbiotinylated DNA does not have the bead joined it and attached to the column. An fluoresces allele specific oligonucleotide is then added to form hybrid with the amplicon. Last but not least, heat is given to determine the Tm of the DNA samples. If there is present of single nucleotide polymorphism the Tm will be lower.


PCR-based methods

In this method, it used two pairs of PCR primers. Each of these primers are specific to their respective alleles.The primer consist of the single nucleotide polymorphorism.Since each pair of primers anneal specifically to their allele. When undergoes PCR process only either one of the primers will bind to the target DNA and amplified.



That all for the week!! Hope your guys have fun and feel free to give comment!!


TG02

Avery, May Lee ( 0503292E)