Tag: Markers

REAL TIME PCR

REAL TIME PCR


Real Time PCR- Principle, Process, Markers, Advantages, Applications

  • Real Time PCR is a technique used to monitor the progress of a PCR reaction in real time.
  • At the same time, a relatively amount of PCR product (DNA, cDNA or RNA) can be quantified.
  • Real Time PCR is based on the detection of the fluorescence produced by a reporter molecule which increases, as the reaction proceeds.
  • A real-time polymerase chain reaction (Real-Time PCR), also known as quantitative polymerase chain reaction (qPCR), is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR).
  • Conventional PCR is a powerful technique that allows exponential amplification of DNA sequences.
  • A PCR reaction needs a pair of primers that are complementary to the sequence of interest. Primers are extended by the DNA polymerase.
  • The copies produced after the extension, so called amplicons, are re-amplified with the same primers leading thus to an exponential amplification of the DNA molecules.
  • After amplification, however, gel electrophoresis is used to analyse the amplified PCR products and this makes conventional PCR time consuming; since the reaction must finish before proceeding with the post-PCR analysis. Real Time PCR overcomes this problem.
  • The term “real time” denotes that it can monitor the progress of the amplification when the process is going on in contrast to the conventional PCR method where analysis is possible only after the process is completed.

Principle of Real Time PCR

This same principle of amplification of PCR is employed in real-time PCR. But instead of looking at bands on a gel at the end of the reaction, the process is monitored in “real-time”. The reaction is placed in to a real-time PCR machine that watches the reaction occur with a camera or detector.

RT-PCR can be done in two ways.

  1. One step RT-PCR
  2. Two step RT-PCR

One step

RT-PCR, both reverse transcription and DNA amplification happen in a single tube. First, at 44°C the ssRNA, specific primers and Reverse transcriptase are used to form dsDNA and then as the temperature is increased to 94°C, the reverse transcriptase is inhibited and the primers and DNA polymerase act to form multiple copies of dsDNA

two step

  • RT-PCR, in the first tube, Reverse transcriptase and non specific primers like oligodTs or random hexamers are used. This step converts any RNA that is present in the sample to dsDNA. This step is carried out at 44°C. This is transferred to another tube into which specific primers, all the four dNTPs and DNA polymerase are added. This is subjected to thermocycling (denaturation at 94°C, Annealing at Tm- 5 and elongation at 72°C) to get multiple copies of DNA.

Advantages of Real Time PCR

  • It gives a look in to the reaction that is help to decide which reactions have worked well and which have failed.
  • The efficiency of the reaction can be precisely calculated.
  • There is no need to run the PCR product out on a gel after the reaction as the melt curve analysis serve the purpose.
  • The real-time PCR data can be used to perform truly quantitative analysis of gene expression. In comparison, old fashioned PCR was only ever semi-quantitative at best.
  • Faster than normal PCR.
  • Less complexity at the quantification of sample.etc

Applications of Real Time PCR

  • Gene expression analysis
    • Cancer research
    • Drug research
  • Disease diagnosis and management
    • Viral quantification
  • Food testing
    • Percent GMO food
  • Animal and plant breeding
    • Gene copy number

As expected the Advantage of Two step RT-PCR is that because the first step uses non specific primers, we can amplify many genes by using more sets of specific primers in the second step. Disadvantage is that it involves multiple steps and hence the workflow is complex and the source of contamination of the sample is higher.

RT-PCR on the whole has few disadvantages -It is not sensitive and not specific. The amplification is not adequate. Requires expensive thermocycling equipment, To overcome these difficulties we use NASBA

NASBA

  • It is a reaction consists of avian myeloblastosis virus (AMV), reverse transcriptase (RT), T7 RNA polymerase and RNase H with two oligonucleotide primers.
  • The steps involved are:
  • The RNA strand present in the sample acts as a sense RNA. To this sense RNA, primer P1 binds (synthesised in such a way that it is complementary to one end of RNA)
  • AMV reverse transcriptase elongates the DNA strand complementary to sense RNA to form RNA:DNA hybrid
  • RNAase H removes RNA part of RNA:DNA hybrid
  • P2 primer binds to the other end of the new DNA strand
  • AMV reverse transcriptase elongates the DNA strand from P2 primer forming a ds DNA
  • P1 primer is so synthesised that once it forms a dsDNA, it acts as a promotor for T7 RNApolymerase. So, once dsDNA is formed, T7 RNA polymerase binds to P1 primer site and it starts transcribing the dsDNA to form multiple copies of RNAs.
  • These antisense RNAs now act as templates for primer P2 and reverse transcriptase acts on it
  • That forms dsDNA and the entire cycle is repeated so that the amplification is more than 1012 fold in 90 to 120 minutes.
  • The whole process happens at 41 °C as there is no need for a denaturation step. Reason being, NASBA uses RNA polymerase and not DNA polymerase

Advantages of NASBA over RT-PCR:

  • The amplification of nucleic acid sequence of more than 109 copies can be done in just 90 minutes by the three-enzyme action.
  • Expensive thermocycling equipment is not needed as the reaction occurs isothermally at 41°C.
  • It helps in better RT-PCR reaction as it offers faster amplification kinetics.

Exam Important

  • Conventional PCR is a powerful technique that allows exponential amplification of DNA sequences.
  • A PCR reaction needs a pair of primers that are complementary to the sequence of interest. Primers are extended by the DNA polymerase.
  • The term “real time” denotes that it can monitor the progress of the amplification when the process is going on in contrast to the conventional PCR method where analysis is possible only after the process is completed.
  • it is used in Disease diagnosis and management, and Viral quantification
  • Zika virus is the example for RT PCR, most sensitive for HIV.
Don’t Forget to Solve all the previous Year Question asked on REAL TIME PCR

Module Below Start Quiz

REAL TIME PCR

Real Time PCR

Q. 1 Real Time PCR is used for:

 A

Multiplication of RNA

 B

Multiplication of specific segments of DNA

 C

Multiplication of Proteins

 D

To know how much amplification of DNA has occurred

Q. 1

Real Time PCR is used for:

 A

Multiplication of RNA

 B

Multiplication of specific segments of DNA

 C

Multiplication of Proteins

 D

To know how much amplification of DNA has occurred

Ans. D

Explanation:

Ans. (d) To know how much amplification of DNA has occured PF 1,m,etz. 25/e 714, 27/e, p 124; Greenwood 18/e 79

Real Time. PCR

  • It is the molecular detection technique that discriminates real time amplification from conventional PCR assays.
    • The real-time polymerase chain reaction (PCR) uses fluorescent reporter molecules to monitor the production of amplification products during each cycle of the PCR reaction.
    • This combines the DNA amplification and detection steps into one homogeneous assay and obviates the requirement for gel electrophoresis to detect amplification products.
    • Its simplicity, specificity, and sensitivity, together with its, more reliable instrumentation, and improved protocols, has made realtime                     oenLhmark technology for lite ocLuLtion of DNA.
    • Real time PCR is extremely useful in medical microbiology, with greatest impact on virology.

Q. 2 Which of the following is most sensitive for diagnosis of HIV?

 A

RT PCR

 B

bDNA assay

 C

NASBA

 D

P24 detection

Q. 2

Which of the following is most sensitive for diagnosis of HIV?

 A

RT PCR

 B

bDNA assay

 C

NASBA

 D

P24 detection

Ans. A

Explanation:

Ans. a. RT PCR


Q. 3

Which of the following techniques is based on RNA?

 A

RT PCR

 B

Sanger’s technique

 C

Next generation sequencing

 D

Western blot

Q. 3

Which of the following techniques is based on RNA?

 A

RT PCR

 B

Sanger’s technique

 C

Next generation sequencing

 D

Western blot

Ans. A

Explanation:

Ans: A. RT PCR
Ref: Harper’s illustrated biochemistry,3Oh ed, pg. 29,457 and Tietz Fundamental of clinical chemistry and molecular diagnostics, Vh ed.
RT-PCR:

  • Reverse transcription polymerase chain reaction (RTPCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase chain reaction (PCR).
  • It is primarily used to measure the amount of a specific RNA. This is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR). Combined RT-PCR and qPCR are routinely used for analysis of gene expression and quantification of viral RNA in research and clinical settings.
  • A method used to quantitate mRNA levels that rely upon a first step of cDNA copying of mRNAs catalysed by reverse transcriptase prior to PCR amplifi cation and quantitation.

 Sanger sequencing,

  • Also known as the chain termination method, is a technique for DNAsequencing based upon the selective incorporation of chain-terminating dideoxynucleotides (ddNTPs) by DNA polymerase during in vitro DNA replication.

Quiz In Between


Q. 4

In which of the following, viral load done by Real Time PCR is of no role in investigative procedures?

 A

Person with hepatitis B on tenofovir therapy

 B HSV causing temporal encephalitis

 C

BK virus in patient of allograft renal transplant

 D

CMV PCR in blood of patient of liver transplant

Q. 4

In which of the following, viral load done by Real Time PCR is of no role in investigative procedures?

 A

Person with hepatitis B on tenofovir therapy

 B

HSV causing temporal encephalitis

 C

BK virus in patient of allograft renal transplant

 D

CMV PCR in blood of patient of liver transplant

Ans. B

Explanation:

Ans. B. HSV causing temporal encephalitis
Ref: “Diagnosis ofherpesvirus infections ofthe central nervous system.’, Herpes : the journal of the IHMF I1 Suppl 2 2004, pp. 48A-564.

  • The question simply asks in which of the given conditions calculation of Viral load is not required. In HSV causing temporal encephalitis the role of PCR is just to detect HSV DNA and make a diagnosis of the disease.
  • There is no role of detection of the viral load copies in the management or diagnosis of the disease.



Q. 5

Best assessment of protein binding regions on a DNA molecule can be done by:

 A

DNA footprinting

 B

RT PCR

 C

Microarray

 D

Western blotting

Q. 5

Best assessment of protein binding regions on a DNA molecule can be done by:

 A

DNA footprinting

 B

RT PCR

 C

Microarray

 D

Western blotting

Ans. A

Explanation:

Answer-(a) DNA footprinting [Ref: www.biotecharticles.com; www.biologyexams4u.com Lippincott 6th/473]

  • DNA footprinting- An in-vitro technique to find out protein binding regions on a DNA molecule. The technique is also called as DNAse I footprinting.Thousands of proteins (enzymes) are interacting with DNA in the nucleus for regulating activities like replication, transcription, translation etc.
  • DNA Footprinting is a molecular technique used to identify the specific DNA sequence (binding site) that binds to a protein.
  • This technique mainly used to identify the transcription factors which bind to promoter, enhancer or silencer region of gene to regulate its expression. Therefore the regulation of transcript ion of a gene can be studied using this method.

Q. 6

True about ZIKA virus:

 A Belong to flavivirus

 B

First case detected in 1953 in Nigeria

 C

RT PCR is useful in diagnosis

 D

Causes macrocephaly

Q. 6

True about ZIKA virus:

 A

Belong to flavivirus

 B

First case detected in 1953 in Nigeria

 C

RT PCR is useful in diagnosis

 D

Causes macrocephaly

Ans. A:C:E

Explanation:

Answer: (a) Belong to flavivirus, (c) RT PCR is useful in diagnosis, (e) May presents with conjunctivitis (Ref: Harrison 19th/1314; www. cdc. gov;www. nytimes. corn] 

  • It is spread mostly by the bite of an infected Aedes species mosquitoes (A. aegptiand A. albopictus). T
  • It can be passed to a pregnant woman to her fetus. Infection during pregnancy can cause certain birth defects..
  • Real-time reverse transcription-polymerase chain reaction (RT PCR) testing should be performed on serum collected during the first two weeks after symptom onset.
  • There’s no vaccine or specific treatment tor tlw disease. Treatment instead focuses on relieving symptoms and includes rest, rehydration and medications for fever and pain.
  • A maculopapular rash, conjunctivitis, myalgia, and arthralgia usually accompany or follow those manifestations.

Quiz In Between



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