Real Time PCR

Ans. (d) To know how much amplification of DNA has occurred 

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 real-time PCR a hallmark technology for amplification of DNA.

Real-time PCR is extremely useful in medical microbiology, with the greatest impact on virology.

Ans. a. RT PCR

Ans: A. RT PCR

Reverse transcription-polymerase chain reaction (RT–PCR) 

• It 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 the analysis of gene expression and quantification of viral RNA in research and clinical settings.
• A method used to quantitate mRNA levels that rely upon the first step of cDNA copying of mRNAs catalyzed by reverse transcriptase before PCR amplification and quantitation.

 Sanger sequencing,

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

Next-generation sequencing:

• Modified methods of genome sequencing. It has significantly reduced the cost and time taken for whole-genome sequencing.
• It is a technique similar to Sanger sequencing, done on DNA 

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.

Answer-(a) DNA footprinting [Ref: http://www.biotecharticles.com; http://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.

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.