PCR is the common type of scientific tool used for amplification of Genomic DNA. Different types of PCR used in labs due to their specificity and sensitivity. This PCR used for the qualitative and quantitative test. PCR Steps are involved de-maturation, annealing, and Extension. Primers, Taq Polymerase, and nucleotides are used.
List of Different types of PCR
- Nested –semi-nested PCR
- Multiplex PCR
- Touchdown PCR
- Inverse PCR
- Allele-specific PCR
- Asymmetric PCR
- Arbitrary PCR
- Core sample PCR
- Degenerate PCR
- Assembly PCR
- Dial-out PCR
- Digital PCR
- Traditional PCR
- Hot start PCR
- In-silico PCR
- Inter-sequence PCR
- Ligation-mediated PCR
- Methylation-specific PCR
- Mini primer PCR
- Nanoparticle PCR
- Overlap-extension PCR
- Quantitative PCR
Most common PCR used
- Simple PCR
- Nested PCR
- q- PRC (quantitative PCR) or Real-Time PCR
- RT – PCR (Reverse transcriptase PCR)
- Multiplex PCR
PCR (Polymerase Chain Reaction)
Polymerase chain reaction involved an essential component
- DNA template
- Primers (reverse and forward)
- Taq Polymerase
Genomic DNA is extracted by using CTAB buffer and Eppendorf is used to collect a sample. In eppendrof extracted DNA and all above ingredients added and put into PCR. Three steps are involved in the Polymerase Chain Reaction.
First Denature Genomic DNA. Double-stranded DNA separated. In Next step Primers Bind to the complementary sequence and in last step Copies are formed.
In each cycle of PCR, the temperature is maintained. In first step 94 – 98 degree. 2nd step 37 -60 degree. And final step about 78 degrees.
Nested PCR is the improvement of polymerase chain reaction was design to improve specificity. It reduces nonspecific binding of Products. Nested PCR used two sets of Primers. One used in the first reaction of polymerase chain reaction and 2nd used in the product of the first reaction to amplifying the purpose
Procedure of Nested PCR
In the first step of nested PCR, target DNA is amplified by using the first set of primers. The Product of the first step amplifies by the 2nd set of primers. 2nd set of primers are complementary of first step product. Purpose of using 2 set of PCR primers is that to reduce contamination of the product and improve its specificity.
Nested PCR image source: Wikipedia
Quantitative PCR is also called real-time PCR. Highly sensitive and reproduce-able technique. This technique could be used quantitatively and semiquantitatively.
Real-Time PCR Principle
It’s basic Principle involved in Thermal cycler. The thermal cycler is an instrument which is linked with PCR that facilitates Thermal cycler Reaction. The common method for detection which is used one is Cyber Green fluorescent dye which Inserts with DNA. 2nd method is Taq man probes of fluorescent labeled is used. This is a laboratory Process. This process consists of amplification by quantitation; Reaction products for each sample on every cycle.
Advantages of Real-Time PCR
- Rapid detection of virus infection like HCV, HBV by using miRNA
- Highly specific to use
- High sensitive
- Reduce Contamination
- Give Quantitation result
- Software-driven operation
Several genomic DNA sequence is amplified in a single step of PCR. Multiple primers and DNA polymerase are used with thermocycler. DNA polymerase is heat mediated. Primers that are used. This PCR is used to detect different pathogens in a single reaction. For Multiplex PCR designing of Primers are Different.
Types of Multiplex PCR
Single Template PCR
To amplify specific sequence of DNA template, this type use single template with multiple forward and reverse primers.
Multiple templates are used with several forward and reverse primers within a single reaction Eppendorf.
Primers of Multiplex PCR
Primers that are used in multiplex PCR design in short length about 18 -22 base pairs.
Advantages of Multiplex PCR
- Highly Efficient than other PCR.
- Amplicon Provide internal control.
Reverse Transcriptase Polymerase Chain Reaction
Reverse Transcriptase PCR is used for cDNA synthesis from RNA. This is also denoted as RT-PCR
But it is different from real-time RCR. That is called q PCR.
Reverse Transcriptase PCR Principle
The first step to form cDNA from RNA by using reverse transcriptase. In Next step amplify this cDNA using PCR. And single-stranded cDNA converted in to double strand DNA. The main point in this whole procedure is the purification of mRNA if mRNA, not purified contamination will occur. This includes 1 step or 2 step method.
In single step method conversion of cDNA and amplification occurred in a single reaction tube and in 2 step method cDNA and amplification proceed in different tubes. 2 step Method consider more convenient than single step method
- Extract RNA
- Extracted material added into Reaction mixture (included reverse Transcriptase, Primers, Target material and nucleotides)
- Primers used to anneal RNA strand if the target is present
- Reverse transcriptase converts mRNA into cDNA
- Primers extend this strand
- The temperature kept 90 degrees to Denature DNA/RNA strand.
- Temperature lowered to anneal the newly formed cDNA.
- New DNA is synthesized by a polymerase enzyme.
- Many copies are formed by multiple cycles.
Application of RT-PCR
- Use to detect genetic Diseases
- Most commonly used to detect RNA virus infection by conversion this into cDNA.
- Used Gene Profiling
- In Future Molecular Diagnosis may be depend on RNA and RT-PCR may be used in coming years.
Applications of PCR (Polymerase Chain Reaction)
PCR is a laboratory Technique used to amplify genomic DNA. In most purpose PCR used. Many types of PCR used for different purpose. As it is used to diagnose diseases, RNA virus infection, Cancer therapy infects in fingerprinting this technique is used. Some important Applications are given below.
- In diagnosis therapy
- n nucleic acid detection assays
- In the medical field
- In agricultural sciences
- In mycology-parasitology
- In dentistry
- In virological diagnostics
- In insert analysis
- In molecular systematic evolution
- In cancer therapy
- In therapy-resistant assessment
- PCR-as biomarker
- In forensic medicine
- In virology
- In bacteriology
- In phytopathology
- In PCR-fingerprinting
- In the detection of microbiological gene
PCR, Nested PCR, Real Time PCR, Reverse Transcriptase PCR, and Multiplex PCR are commonly used in Biochemistry, Biotechnology, Bioinformatics and in labs. Polymerase Chain Reaction amplifies DNA & RNA by making cDNA in reverse transcriptase PCR. This technique is used for qualitative & quantitative purposes.