What Are The Different Types of PCR Techniques?

What are the different tyes of PCR techniques?

PCR is sometimes called “molecular photocopying”. Polymerase chain reaction (PCR) is a quick and inexpensive technique used to “amplify” small segments of DNA. Molecular and genetic analysis requires significant amounts of DNA samples, which makes the study of isolated DNA fragments almost impossible without PCR amplification.

PCR Recognized as one of the most important scientific advances in molecular biology, PCR revolutionized the study of DNA and earned its creator, Kary B. Mullis, the Nobel Prize in Chemistry in 1993.

How many types of PCR are there?

Scientists are performing PCR techniques of many types. however, there are 22 types of PCR types of Techniques that are widely used for DNA amplification.

The list Of PCR techniques Types is as follows.

  1. Real-time PCR
  2. Real-Time Quantitative Reverse Transcription PCR (Q-RT PCR)
  3. Reverse Transcriptase PCR (RT-PCR)
  4. Multiplex PCR
  5. Nested PCR
  6. Long-range PCR
  7. Single-cell PCR
  8. Fast-cycling PCR
  9. Methylation-specific PCR (MSP)
  10. Hot start PCR
  11. High-fidelity PCR
  12. In situ PCR
  13. Variable Number of Tandem Repeats (VNTR) PCR
  14. Asymmetric PCR
  15. Repetitive sequence-based PCR
  16. Overlap extension PCR
  17. Assemble PCR
  18. Intersequence-specific PCR(ISSR)
  19. Ligation-mediated PCR
  20. Miniprimer PCR
  21. Solid phase PCR
  22. Touchdown PCR
What are the different types of PCR technuques?

1. Real-time PCR

Real-time PCR is commonly used to measure gene expression. It is more sensitive than microarrays for detecting small changes in expression, but requires more input RNA and is less suitable for high-throughput studies.

Ideal for studying small subsets of genes. One of its major drawbacks is that the sequence of the specific target gene must be known (so that PCR primers can be designed). Therefore, real-time PCR can only be used to study known genes.

2. Real-Time Quantitative Reverse Transcription PCR (Q-RT PCR)

Real-time Quantitative reverse transcription-PCR is a major advance in PCR technology that allows reliable detection and measurement of the products produced in each cycle of the PCR process.

This technology became possible after the introduction of oligonucleotide probes designed to hybridize to the target sequence. Probe cleavage during PCR by the 5′ nuclease activity of Taq polymerase can be used to detect amplification of a specific target product.

Q-RT PCR is used for relative and absolute quantification of gene expression, validation of DNA microarray results, variation analysis including SNP discovery and validation, bacterial, viral or fungal load enumeration, etc.

3. Reverse Transcriptase PCR (RT-PCR)

Reverse transcription PCR (RT-PCR) is a variant of conventional PCR in which RNA molecules are first converted into complementary DNA molecules (cDNA) and then amplified by PCR.

In RT-PCR, an RNA template is first converted into complementary DNA (cDNA) using reverse transcriptase. The cDNA then serves as a template for exponential amplification using PCR.

RT-PCR can be performed in one tube or in two steps in different tubes. The one-step method is more efficient and involves less potential contamination and variability.

RT-PCR is used in research methods, gene insertion, diagnosis of genetic diseases and cancer diagnosis.

4.  Multiplex PCR

Multiplex PCR is the simultaneous detection of multiple targets in one reaction well using different primer pairs for each target. This technique requires two or more probes that can be distinguished from each other and detected simultaneously. A variety of probe technologies are available, all using fluorophores.

Multiplex PCR is used in life science research, clinical diagnostics and forensic laboratories. The development of PCR detection systems that detect multiple targets simultaneously and advances in probe chemistry have standardized comparative analysis in many areas of research and testing.

The most common applications of Multiplex PCR include SNP genotyping, pathogen detection, GMO (genetically modified organism) detection, forensic investigation, food analysis, mutation and polymorphism analysis, gene deletion analysis, linkage analysis, RNA detection.

5. Nested PCR

Nested PCR usually involves two consecutive amplification reactions, each using a different primer pair. The product of the first amplification reaction is used as a template for a second PCR primed by an oligonucleotide inserted into the first primer pair.

Nested PCR is a useful modification of PCR technology that increases the specificity of the reaction by using two sets of primers to prevent non-specific binding.

The first set of primers binds to the outside of the target DNA and amplifies larger fragments, while another set of primers bind specifically to the target site. In the second round of amplification, the second set of primers amplifies only the target DNA.

Nested PCR is a useful method for phylogenetic studies and detection of various pathogens. This technique is more sensitive. Therefore, even low-level DNA in the sample can be amplified, which is not possible with conventional PCR techniques.  

6. Long-range PCR

Long-range PCR refers to the amplification of DNA targets greater than 5 kb in length that typically cannot be amplified by standard PCR methods and reagents.

Long-range PCR is a method for amplifying longer lengths of DNA that cannot normally be amplified with standard PCR methods and reagents.

Long-range PCR can be achieved using modified high-performance polymerases with enhanced DNA binding, which results in highly consistent and accurate amplification of long fragments.

This method allows amplification of a more wider range of goals in a shorter period of time and efficient use of resources.

7. Single-cell PCR

A high level of heterogeneity in gene expression profiles is observed among cells of the same tissue or cell population. Even cells with the same apparent phenotype can show wide variation in the set of expressed genes and their expression levels.

Single-cell analysis can be used to study the profiles of different cell types in tissues, the diversity of cells in populations, and differences in cellular processes such as differentiation and response to stimuli.

Using qPCR for single-cell assays is technically challenging due to the small amount of nucleic acids present in a single cell. Digital PCR can overcome several barriers to single-cell analysis by increasing sensitivity and absolute quantification.

8. Fast-cycling PCR

Rapid PCR enzymes reduce cycle times, resulting in faster sample-to-results and higher throughput. Phusion and Phire DNA polymerases incorporate more nucleotides per splicing event than other polymerases. The above process allows the use of very short amplification times, resulting in very short overall PCR cycles.

9. Methylation-specific PCR (MSP)

Methylation-specific PCR (MSP) is a method for analyzing the DNA methylation pattern of CpG islands. To perform MSP, DNA is modified with two pairs of primers, respectively, detectably methylated and unmethylated DNA and PCR is performed. MSP is a rapid tool to assess the methylation status of CpG islands.

The basic principle of MSP is the specific PCR amplification of bisulfite-converted DNA. Treatment of genomic DNA with sodium bisulfite converts unmethylated cytosines in a DNA sample to uracil, whereas methylated cytosines are resistant to this modification and remain unchanged.

10. Hot start PCR

Hot-start PCR is a convenient technique that reduces non-specific amplification and allows reactions to be set up at room temperature. Polymerases used in hot-start PCR do not react at room temperature.

Polymerase activity can be inhibited at these temperatures through various mechanisms such as antibody interaction, chemical modification, and aptamer technology. At the permissive reaction temperature reached during the PCR cycle, the polymerase dissociates from its inhibitor and starts polymerization.

The use of hot-start DNA polymerases is often recommended for high-throughput applications, experiments that require a high degree of specificity, or routine PCR that requires the additional security provided by hot-start enzymes.

11. High-fidelity PCR

High-fidelity PCR enzymes are used for applications that require high precision during DNA replication, such as cloning, sequencing, and mutagenesis. Thermo Scientific Phusion High Fidelity DNA Polymerase was created by fusing a dsDNA binding domain to a Pyrococcus polymerase-like proofreading enzyme.

Due to this unique protein fusion engineering, Phusion DNA polymerases have very low error rates, fast elongation rates, and high inhibitory resistance.

12. In situ PCR

In-situ PCR (In-situ PCR) is an effective method for identifying small amounts of rare nucleic acid sequences in cells or frozen or paraffin-embedded tissue sections and segmenting intracellular sequences.

This method involves tissue fixation that preserves cell morphology, followed by treatment with proteolytic enzymes to provide an entry point for PCR reagents to act on the target DNA.

Target sequences are amplified by reagents and detected by standard immunocytochemistry protocols.

In situ PCR can also be used for the diagnosis of infectious diseases, DNA quantification, and detection of low abundance DNA, and is widely used to study organogenesis and embryogenesis.

13. Variable Number of Tandem Repeats (VNTR) PCR

Variable number tandem repeats (or VNTRs) are locations in the genome where short nucleotide sequences are organized as tandem repeats. They are found on many chromosomes and often vary in length (number of repeats) between individuals.

Each variant acts as an inherited allele and can be used to identify an individual or a parent. Their analysis is useful in genetics and biology research, forensics and DNA fingerprinting.

14. Asymmetric PCR

Asymmetric PCR is a type of PCR that is used to preferentially amplify one strand of the original DNA over another. This technique is applicable to several types of sequencing and hybridization probes that require only one of the two complementary strands.

Asymmetric PCR can be used to make single-stranded DNA from double-stranded DNA. It is used to determine DNA sequence in mutagenesis methods. Single-stranded DNA is also important for aptamer generation.

15. Repetitive sequence-based PCR

PCR based on the sequence of repetitive elements (rep-PCR) is a typing method that allows the generation of DNA fingerprints for the identification of bacterial strains.

Repeat element sequence-based PCR (rep-PCR) isolates microorganisms using primers complementary to interspersed repetitive consensus sequences that allow the amplification of DNA fragments of various sizes consisting of sequences between repeat elements. This is a new way to type for differentiation.

16. Overlap extension PCR

Overlap extension polymerase chain reaction (or OE-PCR) is a variant of PCR. Also known as Splicing by overload extension/Splicing by overhang extension (SOE) PCR. It is used to introduce specific mutations at specific positions in a sequence or to connect small pieces of DNA to larger polynucleotides.

17. Assemble PCR

PCR assembly using synthetically derived DNA is a flexible technique for generating new gene sequences. Generate long genes up to thousands of base pairs using single-stranded or mixed single- and double-stranded DNA oligos. This approach is also useful when making constructs using modular elements such as antibodies.

PCR assembly is also interesting because overlapping sequences can be joined together without the need for restriction sites and robust PCR reagents and methods are available. However, PCR assembly usually requires combining many short fragments, which requires careful planning and extensive optimization for a successful experiment.

18. Intersequence-specific PCR(ISSR)

Inter-simple sequence repeat (ISSR)-PCR is a technique that uses microsatellite sequences as primers for the polymerase chain reaction to generate multiple locus markers.

It is a simple and rapid method that combines most of the advantages of microsatellites (SSR) and amplified fragment length polymorphism (AFLP) with the ubiquity of randomly amplified polymorphic DNA (RAPD).

ISSR markers are highly polymorphic and useful in studies of genetic diversity, phylogeny, gene tagging, genome mapping, and evolutionary biology. The sequences amplified by ISSR-PCR can be used for DNA fingerprinting.

19. Ligation-mediated PCR

Ligation-mediated PCR (LM-PCR) is a classic method for isolating flanking sequences. However, there is a common limitation that circularization or polymerization of target restriction fragments containing known sequences reduces the success rate.

20. Miniprimer PCR

Mini-primer PCR amplified a greater proportion of novel sequences or sequences with poor matches to previously isolated 16S rRNA gene sequence databases than standard primers. It confirmed predictions based on computer searches of the NCBI environmental sequence database.

21. Solid phase PCR

Solid-phase PCR (SP-PCR) is of interest in various research fields because it enables parallel amplification of DNA on the surface of solid substrates. However, the use of SP-PCR is hampered by the low efficiency of solid phase amplification.

22. Touchdown PCR

“Touchdown polymerase chain reaction (PCR)” is a method to reduce off-target priming and increase the specificity of PCR. In touchdown PCR, the temperature selected for the annealing step is initially set 5°C to 10°C higher than the calculated Tm of the primers.

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