TYPE OF HYDROLYSIS PROBE

TYPE OF HYDROLYSIS PROBE

17.      TYPE OF HYDROLYSIS PROBE

17.1.     TaqMan probe / molecular probe / fluorescence probe : It is an oligonucleotide of 20-26 bases, it binds only with the target DNA sequence and contains fluorochrome (donor) at 5’end along with quencher–accepter (TAMRA) at 3’end, thus no fluorescence observe when both present at Taqman probe because it is work on FRET. 
During the extension, fluorochrome–donor (FAM, GFP, TET) at 5’end get removed by 5’-3’exonuclease activity of the polymerase, as a result, detection takes place in extension phase, here fluorochrome is attached with base thus gets recognized inappropriate by the polymerase and gets cleaved. This is specifically used in the multiplex assay.

17.2.     Molecular beacons: It is an oligonucleotide having the stem-loop structure with fluorochrome (donor) at 5’end along with quencher–accepter at 3’end at the ends of stem-loop. 

When Molecular beacons are present its stem-loop structure then no fluorescence observed due to FRET, but when the temperature increases beacon gets extended. In each denaturation cycle, the same fluorescence intensity is observed so we can not identify that the product is formed or not, like that in each extension same fluorescence intensity is observed. 
But in annealing stage fluorescence increment takes place gradually as increase in number of template increase with each cycle, because probe get hybridize with template in extended form and detection takes place simultaneously increase with the product and when fluorescence of annealing get equalize with denaturation and extension, the time comes when machine need to close as well as reaction get complete.

17.3.     Scorpion probe: It is probe along with primer, here a stem-loop or hairpin loop probe attached to 5’ end of primer through the linker, stem-loop structure contain fluorochrome (donor) at 5’ end along with quencher (accepter) at 3’end at the ends of stem-loop and quencher attached to the linker. 
After or during the extension, probe hybridization region is formed within extended amplicon, as a result, the probe gets hybridized with its complementary region by the opening of hairpin loop and signal observe in extension.
17.4.    RT-PCR (Reverse transcriptase PCR)
This type of PCR uses mRNA or RNA as an initial sample. mRNA or RNA firstly convert it to cDNA by reverse transcriptase enzyme and then amplification of this cDNA takes place.
Isolation of mRNA from the cell is done by affinity chromatography through the deoxdeoxythymidineand. After that conversion of mRNA into cDNA occurs in first strand reaction and cDNA amplification occur in second strand reaction. This whole process is carried out in one tube, for the first reaction, deoxythymidine act as a primer for reverse transcriptase and formation of ss cDNA takes place as reverse transcriptase synthesize the cDNA and simultaneously degrade the mRNA which acts as the template for cDNA synthesis. After that this cDNA is converted into ds cDNA with the help of reverse transcriptase enzyme by using ss cDNA act as a template and the non hybridized ss cDNA present is between ds cDNA is cleaved by S1 endonuclease. This reaction is carried out at 37°C with the help of reverse transcriptase. The reverse transcriptase use in both reactions. name of some other reverse transcriptase, AMV (Avian Myeloblastosis Virus) reverse transcriptase, Mo-MLV (Moloney Murine Leukemia Virus) reverse transcriptase.


Now the second reaction takes place in which deoxy thymidine act as one primer and we need a second primer which is specific for cDNA and made by 5’UTR of mRNA because it is complementary to 5’UTR of mRNA. ~35 cycles are required to carry out amplification of cDNA. cDNA is used to form cDNA libraries and diagnosis of genetic diseases. 
17.5.    Hot start PCR
In the hot start, PCR polymerization activity of the polymerase is inhibited prior to first denaturation cycle because all reaction mixture is present within a reaction tube with a polymerase which causes non-specific DNA synthesis due to non-specific binding of primer. Thus in hot start, PCR addition of DNA polymerase takes place after the first cycle DNA denaturation step at the temperature above or same as annealing temperature just previously to the first cycle annealing step. Hot start PCR is used specifically in multiplex PCR.
17.6.    Nested PCR
In this type of PCR, two sets of primer use, in which the first set of primer is used to carry out standard PCR reaction. After that second PCR reaction is carried out by the second set of primer or internal primer whose site is just internal or downstream to the site of the first set of primer but complementary to the target. Internal primer uses the product of the first reaction as the template which strictly reduces the formation of a non-specific product which is produced in the course of the first reaction. This PCR is specifically used in nick translation and increases the specificity of the PCR product.

17.7.    Inverse PCR or Inside out PCR or Inverted PCR
If the primer is not available for target DNA sequence and only the sequence present in the middle of the target DNA sequence is known then the PCR reaction is carried out in the following manner. The target sequence flanking the known internal sequence from both sides should possess the restriction site of same restriction enzyme name RE1 and known internal or middle sequence also possess restriction site for other restriction enzyme name RE2. Prime for PCR reaction is made by the information of known internal sequence.  
Firstly restriction digestion with RE1 is carried out which result in the cohesive site at both ends of target DNA, thus DNA gets circularized and now restriction digestion with RE2 is carried out which results in linear DNA with the known sequence at both ends as well as a primer for both ends also available. As a result PCR reaction is carried out and amplification of sequence occurs. When the PCR reaction completes, then linear DNA again gets circularized because the RE2 restriction site also produces cohesive end resulting in circular DNA. After that restriction digestion with RE1 is again carried out which result in the production of the original target DNA sequence with amplification. This PCR use in identification of flanking sequence present around genomic insert as well the recognition of genomic insert. It is used in chromosome walking for the production of the end-specific probe.


Anchored PCR
In the basic PCR technique and the inverse PCR, one has to use two primers representing the sequences lying at both ends of the sequence to be amplified. But sometimes, we may have knowledge about the sequence at only one of the two ends of the DNA sequence to be amplified. In such cases anchored PCR may be used, which will utilize only one primer instead of two primers. In this technique, due to the use of one primer, only one strand will be copied first, after which a poly G tail will be attached at the end of the newly synthesized strand. This newly synthesized strand with poly G tail at its 3'-end will, then, become the template for the daughter strand synthesis utilizing an anchor primer with which a poly C sequence is linked to complement with poly G of the template. In the next cycle, both the original primer and anchored primer will be used for gene amplification.


17.8.    Asymmetric PCR
Asymmetric PCR is used when there is a need to amplify one DNA sequence preferentially from the other DNA sequence within ds DNA. This is accomplished by the use of one primer in excess concentration than other primers, which is specific for the DNA sequence which needs more amplification.
17.9.    Allele-specific PCR
If we need to amplify a specific allele then we need to make primer according to that allele, in turn, cause the production of a particular allele.
17.10.    VNTR PCR
In this type of PCR, (variable number of Tandem repeats) VNTR flank the target gene.
17.11.    Internal or Inter SSR PCR
In this type of PCR SSR (simple sequence repeat) flank the target gene.
17.12.    Random amplified polymorphic DNA (RAPD) or AP-PCR (Arbitrary primed PCR)
It is another technique based on PCR. It is used to compare the PCR amplification profile of different sample collected from the same place or different place in one PCR reaction. This is specially usd in forensic science to amplify the different sample received from crime place in one PCR reaction. This technique, in turn, detect polymorphism among different sequence This task is accomplished because in this PCR arbitrary primer of 8-12 nucleotide is used, but mostly decamer is use and DNA synthesis carried out by Taq DNA polymerase.
In this technique large target DNA sequences are used which may possess two or more binding site for arbitrary primer and if primer hybridize the place which allows the target strands amplification from both places then it get amplify from both place thus polymorphism can be shown between different sequence because both primers had different binding site one each strand. Thus it acts as a dominant genetic marker and uses to identify the polymorphism in the phylogeny among diverse animal and plant species.
Steps like denaturation, annealing and extension in RAPD are the same as in standard PCR but here cycle repetition occurs 40 -50 times. In RAPD more than 128 primers are used. Results are seen on the gel after the completion of the reaction. EtBr is used to visualize the bands on the gel. Here previous knowledge of the target DNA sequence is not required. RAPD in not able to differentiate the heterozygousity or homozygousity between amplified DNA sequences. 
17.13.    Restriction fragment length Polymorphism (RFLP)
RFLP is a hybridization based molecular marker. The mutation in the restriction site causes the generation of different length of the fragment from the same genomic segment after the digestion with the same restriction enzyme. Thus by RFLP,  we can define the difference in length between two chromosomes which arise by single mutation within the restriction site. As a result, we can define the hetrozygousity by RFLP and 0.5 is the value of maximum heterozygosity. In one time RFLP work on one locus. RFLP is highly reproducible and a codominant marker. [RFLP is better than RAPD. Through RFLP we can differentiate between homozygote and heterozygote.]
Steps involve in RFLP as follows: DNA from the different subject is isolated which is thought to show polymorphism then restriction digestion is performed by restriction enzyme (one or more). After that resulted fragment is subjected to agarose gel electrophoresis and then the gel is subjected to Southern blotting. Now bands of DNA are immobilized on the membrane after that hybridization of radiolabeled probe takes place and the result can be visualized by autoradiography.

Amplified Fragment Length Polymorphism
This technique involves PCR amplification selectively for restriction fragment generated by the digestion of genomic DNA by two restriction enzyme. This technique is highly reproducible and highly sensitive for detecting polymorphism in genomic DNA, better then RAPD and RFLP. AFLP is the dominant marker.
The procedure of AFLP include the following steps, firstly isolation and purification of genomic DNA take place, then genomic DNA is subjected to restriction digestion with the help of two restriction enzyme, in which one restriction enzyme are frequent four base cutter Mse I and other is rare six base cutter Eco RI. After that resulted restriction fragment is ligated with the restriction enzyme specific adaptor at the particular site where cut by particular restriction enzyme and ligate with the help of T4 DNA ligase. After that first PCR amplification was done with the help of primer which is the complement to the restriction site of the restriction enzyme along with single nucleotide at 3’ end of the primer and with this primer 20 cycles of PCR reaction carried out which involves all standard PCR cycle steps. 
After that second PCR reaction is carried out with the primer which possesses 3 selective nucleotides at 3’ end in spite of one which is present in the first reaction primer. One primer from these two primers end is radiolabeled with polynucleotide kinase and (γ32P). This radiolabeled primer is specifically Eco RI primer. Second PCR reaction is performed up to 36 PCR cycles which involves all standard PCR cycle steps. Resulted amplified fragment are separated with the help of denaturing polyacrylamide gel. Then bands on the gel are identified by autoradiography and polymorphism is detected. Silver staining or fluorescence probe also can be used if the radiolabeled nucleotide is not used.


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