Labtory Methods- Molecular

What is polymerase Chain reaction?

• Method to identify low frequency variants precisely even with low-quality or limited-input samples

• Highly efficient method to amplify low levels of specific DNA sequences in a specimen to reach threshold of detention.

• highly efficient method that offers a complete service in the analysis of genetic material.

• method that determines the genome sequencing of an organism

Answer explanation

- Two short DNA "primers“ = oligonucleotides (small portions of a single DNA strand) specific for viral DNA sought - sequence flanks that section of DNA to be amplified

- Repeated cycles of DNA denaturation (separation of double DNA strands), primer annealing (recombination of double-stranded structure) & extension of the primed DNA sequence.

What is the principle of PCR?

• Amount of amplified product determined by available substrates.

• Amplyfies a DNA strand to code a specific genome.

• used to amplify a specific region of a DNA strand.

• Used to amplify a specific region of a RNA strand.

How does Real-Time PCR detection work?

• Fluorescent molecules added to PCR reaction mix.

• Fluorescent and PCR mixture interact with PCR product

• increase in fluorescent signal when PCR amplification occurs.

• All of the above

Answer explanation

-it can readily be adapted to quantify amount of RNA or DNA in specimens.

-Real-time PCR achieved by use of fluorescent detection technology.

What is principle of quantitative PCR?

• - During thermo-cycling amplification begins sooner in specimen with lower viral load only

• - during thermo-cycling amplification begins sooner in specimens with higher viral load compared with specimens with lower viral load.

• - during thermo-cycling amplification begins sooner in specimens with higher viral load

• none of the above

Limitations of molecular methods are:

• -Conversely, inhibitors in specimens or nucleic acid degradation may lead to false-negative results.

• -If multiple copies of DNA from a laboratory contaminant or previous experiment are present, then a false-positive result will occur

• -Molecular tests using nucleic acid amplification has many disadvantages – exercise caution when using these tests.

• all of the above

Answer explanation

-Molecular tests using nucleic acid amplification has many advantages – exercise caution when using these tests.

- If only one copy of DNA from a laboratory contaminant or previous experiment is present, then a false-positive result will occur

select the correct sequence of Hybrid Capture form below:

1. Each hybrid combines with many Ab-Enzyme molecules resulting in signal from amplification

2. Captured hybrids detected by a secondary Ab attached to an enzyme

3. captured onto solid phase by Abs specific to RNA:DNA hybrids

4. cleaves chemiluminescent substrate →light signal

5. Target DNA & specific RNA probes combine

6. RNA:DNA hybrid

• 3-5-2-6-1-4

• 5-6-3-4-2-1

• 2-4-3-5-1-6

• 5-6-2-4-3-1

What are the 3 enzymes used by NASBA?

• - RNase H

  - T3 RNA polmerase

  - ECL polymerase

• -T7 RNA polymerase

  - Primer 2

  - RNase P

• -Avium Myeloblastosis virus Reverse Transcriptase (AVM-RT)

  -T7 RNA polymerase

  -RNase H

• -Avium Myeloblastosis virus Reverse Transcriptase

  - T7 promoter

  - Primer 1

Answer explanation

-NASBA amplifies RNA from an RNA target using a dual function reverse transcriptase

-Based on amplification of a target sequence by in-vitro transcription

List the enzymes used in Transcription-Mediated Amplification (TMA).

• -Reverse transcriptase

• - RNA polymerase has Reverse transcriptase & RNaseH activity

• - Promoter- primerase

• All of the above

Answer explanation

-Uses ONE enzyme - RNA polymerase has Reverse transcriptase & RNaseH activity → RNA transcription

-Target nucleic acid → RNA amplification product → reverse transcriptase → DNA synthesis

-Possibility of carryover contamination ↓ - RNA more labile compared to DNA

rolling circle amplification is best descripbed as ...

• Modification of RCA - use two primers = exponential, hyper branched, ramification, or cascade RCA

• Polymerase displaces upstream sequences to long single stranded DNA containing multiple repeat copies of target sequence

• All of the above

• Amplification of a circular target molecule by extension of a single forward primer by DNA polymerase for many rounds.