rDNA

Types of PCR and Microarrays

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By : Sumit Sharma

Department of Biotechnology

Types of PCR

• Multiplex PCR
• Nested PCR
• RT-PCR and qRT-PCR
• Quantitative PCR
• Hot-star PCR
• Touchdown PCR
• Assembly PCR
• Colony PCR
• Methylation –specific PCR
• Lamp assay

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Quantitative PCR

• It is used to measure the specific amount if target DNA in a sample .
• By measuring amplification only with in the phase of true exponential increase , the amount of measured product more accurately reflects the initial amount of target .
• Special thermal cyclers are used that monitor the amount of product during the amplification.

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Hot –star PCR

It is a technique performed manually by heating the reaction components to the DNA melting temperature before adding the polymerase.

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Touchdown PCR

• In this annealing temperature is gradually decrease in later cycle.
• The annealing temperature in the early cycle is usually 3-5 °C above the standard temperature of the primers used, while in later cycle it is below.
• The initial higher annealing temperature leads to greater specificity for primer binding , while the lower temperatures permit more efficient amplification at the end of the reaction

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Assembly PCR

• This technique is substitute for Ligation-based assembly.
• This type involves the synthesis of long DNA structure by performing PCR on a pool of long oligonucleotides with short overlapping segments , to assemble two or more pieces of DNA into one piece.
• It involves an initial PCR with primers that have an overlap and a second PCR using the products as the template that generates the final full length product.

Colony PCR

• Bacterial colonies are screened directly by PCR , e.g. the screen for correct DNA vector constructs
• Colonies are sampled with a sterile pipette tip and small quantity of cells transferred into PCR mix

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Methylation specific PCR (MSP)

• Used to identify patterns of DNA methylation at cytosine guanine in genomic DNA .
• Target DNA is first treated with sodium bisulfate , which convert unmethylated cytosine bases to uracil , which is complementary to adenosine in PCR primers
• Two amplification are then carried out on the bisulfate treated DNA
• One primers set anneals to DNA with cytosine
• It is used in quantitative PCR provides quantitative information about the methylation state of a given CpG

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LAMP assay

• LAMP assay stands for Loop mediated type isothermal amplification
• It is a modified type of PCR using 3: 6 primers sets one of them is loop like primer
• This test use Bst- polymerase enzyme
• Use of only two temperature i.e. 63 °C for 5 min. then 85 °C for 5 min. , may be carried out in water path

Application

• Detection of mutation ( investigation of genetic diseases)
• Cloning genes
• PCR sequencing
• Identification and characterization of infectious agents
• Direct detection of microorganisms in patient specimens
• Identification of microorganisms grown in culture
• Detection of antimicrobial resistance
• Investigation of strain relatedness of pathogen of interest

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Advantages

• This technique used for producing billons of copies of specific product for sequencing , cloning , and analysis
• Used to analyze alteration of gene expression levels in tumors , microbes or other disease state.
• PCR is very powerful and practical research tool .
• Help to identify the sequence of previously unknown viruses

Limitation

• If the gene is unknown , we cannot make the appropriate primer.
• PCR is very sensitive technique , it may generate false signals due to the previous contamination
• Amplicon size is very important

Microarray

A microarray is a pattern of ssDNA probes which are immobilized on a surface (called a chip or a slide). The probe sequences are designed and placed on an array in a regular pattern of spots. The chip or slide is usually made of glass or nylon and is manufactured using technologies developed for silicon computer chips. Each microarray chip is arranged as a checkerboard of 105 or 106 spots or features, each spot containing millions of copies of a unique DNA probe

Contd..

Like Southern & northern blots, microarrays use hybridization to detect a specific DNA or RNA in a sample. But whereas a Southern blot uses a single probe to search a complex DNA mixture, a DNA microarray uses a million different probes, fixed on a solid surface, to probe such a mixture. The exact sequence of the probes at each feature/location on the chip is known. Wherever some of the sample DNA hybridizes to the probe in a particular spot, the hybridization can be detected because the target DNA is labeled (and unbound target is washed away). Therefore one can determine which of the million different probe sequences are present in the target.

Principle

Microarray is the hybridization between two strands of DNA

Fluorescent labeled target sequences that bind to a probe sequence generate a signal that depends on the strength of the hybridization determined by the number of paired bases.

References

General Biotechnolgy by BD Singh

Gene XI by Benjimin Lewin