ASCP Technologist in Molecular Biology 2 — Questions and Answers
Question 1: What is the principle behind polymerase chain reaction (PCR)?
- Separating proteins by size
- Amplifying specific DNA sequences through repeated cycles of denaturation, annealing, and extension (Correct answer)
- Culturing bacteria in specialized media
- Measuring antibody-antigen reactions
Correct answer: Amplifying specific DNA sequences through repeated cycles of denaturation, annealing, and extension
PCR amplifies specific DNA sequences exponentially through thermal cycling: denaturation, annealing, and extension.
Each PCR cycle: (1) Denaturation at 94-98 degrees C separates double-stranded DNA, (2) Annealing at 50-65 degrees C where primers bind to target sequences, (3) Extension at 72 degrees C where Taq polymerase synthesizes new strands. Each cycle doubles the target, so 30 cycles produce over 1 billion copies. PCR is used for infectious disease detection, genetic testing, and forensics.
Question 2: What is the difference between real-time PCR (qPCR) and conventional PCR?
- qPCR is faster but less accurate
- qPCR monitors amplification in real-time using fluorescent probes, enabling quantification (Correct answer)
- Conventional PCR uses higher temperatures
- No significant difference
Correct answer: qPCR monitors amplification in real-time using fluorescent probes, enabling quantification
Real-time PCR uses fluorescent reporters to monitor amplification during each cycle, enabling quantification of starting template.
qPCR uses fluorescent systems (TaqMan probes, SYBR Green) to measure amplification each cycle. The Ct value (cycle number where fluorescence exceeds background) is inversely proportional to starting DNA amount. qPCR is essential for viral load monitoring (HIV, HCV), minimal residual disease detection, and COVID-19 testing.
Question 3: What is the purpose of reverse transcriptase in RT-PCR?
- To denature DNA at high temperatures
- To convert RNA into complementary DNA (cDNA) before PCR amplification (Correct answer)
- To separate DNA fragments by size
- To label DNA with fluorescent markers
Correct answer: To convert RNA into complementary DNA (cDNA) before PCR amplification
Reverse transcriptase synthesizes cDNA from an RNA template, enabling RNA viruses and gene expression to be detected by PCR.
RT-PCR is a two-step process: reverse transcriptase converts RNA to cDNA, then standard PCR amplifies it. Essential for detecting RNA viruses (SARS-CoV-2, HIV, HCV), measuring gene expression (mRNA levels), and detecting fusion gene transcripts in cancer (BCR-ABL in CML). RT-qPCR combines both techniques.
Question 4: Which molecular technique detects specific DNA sequences by separating fragments and transferring them to a membrane?
- Northern blot
- Southern blot (Correct answer)
- Western blot
- ELISA
Correct answer: Southern blot
Southern blotting separates DNA fragments by gel electrophoresis, transfers to a membrane, and hybridizes with a labeled probe.
Southern blotting: DNA digestion with restriction enzymes, gel electrophoresis separation, transfer to nylon membrane, hybridization with labeled complementary probe, and detection. While largely replaced by PCR, it remains useful for gene rearrangements and trinucleotide repeat expansions. Northern blot detects RNA; Western blot detects proteins.
Question 5: What is next-generation sequencing (NGS) and how has it impacted clinical diagnostics?
- A faster version of Sanger sequencing
- Massively parallel sequencing of millions of DNA fragments simultaneously (Correct answer)
- A protein sequencing technique
- A method for growing bacteria quickly
Correct answer: Massively parallel sequencing of millions of DNA fragments simultaneously
NGS performs massively parallel sequencing of millions of fragments, enabling comprehensive genomic analysis at unprecedented speed.
Major platforms include Illumina (sequencing by synthesis), Ion Torrent, and Oxford Nanopore. Clinical applications include whole genome sequencing, cancer panels (precision medicine), metagenomic sequencing (pathogen identification without culture), pharmacogenomics, and non-invasive prenatal testing. Costs have dropped from $100 million to under $1,000 per genome.
Question 6: What is FISH (fluorescence in situ hybridization) used for?
- Growing fluorescent bacteria
- Detecting specific DNA/RNA sequences in cells using fluorescent-labeled probes (Correct answer)
- Measuring body fluid pH
- Performing automated blood counts
Correct answer: Detecting specific DNA/RNA sequences in cells using fluorescent-labeled probes
FISH uses fluorescent-labeled DNA probes that hybridize to specific chromosome regions, enabling visualization of chromosomal abnormalities.
FISH detects chromosomal translocations in leukemia (BCR-ABL in CML), gene amplification in tumors (HER2/neu in breast cancer determining trastuzumab eligibility), microdeletions (DiGeorge syndrome), aneuploidies (rapid prenatal trisomy diagnosis), and engraftment monitoring after bone marrow transplant.
ASCP Technologist in Molecular Biology 2
What is the principle behind polymerase chain reaction (PCR)?