LABORATORY TECHNIQUES IN IMMUNOLOGY AND SEROLOGY (Molecular Techniques) Flashcards
memorization
He invented the polymerase chain reaction (PCR) in 1983 and was awarded the Nobel Prize in 1993
Kary Mullis
Components of PCR:
- Thermostable DNA polymerase
- Deoxynucleotides of each base
- DNA containing target sequence
- Oligonucleotide primers (20-30 nucleotides long)
Temperature requirement in denaturation step:
95 degrees Celsius
Temperature requirement in primer binding or annealing
52 degrees Celsius
At ____ degrees Celsius, polymerase binds to the 3’ end of the primer and synthesizes a new DNA strand
72 degrees Celsius
The amplified DNA sequences are called:
Amplicons
If the nucleic acid of interest is RNA, it can be converted to DNA in the initial step of PCR by using:
Reverse transcriptase
A PCR technique that is useful in identification of HIV and Hepatitis C; also a confirmatory test for HIV and Hepa C
Reverse Transcriptase Polymerase Chain Reaction (RT PCR)
A PCR technique that uses numerous primers within a single reaction tube to amplify nucleic acid fragments from different targets
Multiplex Polymerase Chain Reaction (MPCR)
A PCR technique that measures the amount of amplicons in real-time, which reduces the time it takes to run a PCR; less susceptible to amplicon contamination
Real-Time Polymerase Chain Reaction or Quantitative PCR (qPCR)
Blotting procedure where DNA is denatured with restriction enzymes to create DNA fragments
Southern blot
Blotting procedure where mRNA is separated by electrophoresis and blotted
Northern blot
Blotting procedure where proteins are separated electrophoretically, and identified through the use of labeled antibodies specific for the protein of interest
Western blot
Which is associated only with RNA synthesis?
a. Promoter
b. Cytosine
c. S phase
d. Primer
Option a: Promoter
Promoters are specific DNA sequences where RNA polymerase binds to initiate transcription (RNA synthesis). They play a crucial role in regulating gene expression.
Options clarification:
1. Option b: Cytosine - DNA/RNA base.
2. Option c: S phase - DNA replication phase.
3. Option d: Primer - DNA/RNA synthesis initiation (both PCR and DNA replication).
The speed at which nucleic acids migrate in gel electrophoresis is determined by which property?
a. Charge
b. Fluorescence
c. Absorption
d. Size
Option d: Size
Gel electrophoresis separates nucleic acids (DNA/RNA) based on:
Key Factors
1. Size: Smaller fragments migrate faster.
2. Charge: Negatively charged phosphate backbone (uniform in nucleic acids).
3. Shape: Linear, circular, or supercoiled.
Electrophoresis Principles
1. Electric field applies force.
2. Negatively charged nucleic acids move toward positive electrode.
3. Gel matrix retards larger fragments.
Techniques
1. Agarose gel electrophoresis (DNA separation).
2. Polyacrylamide gel electrophoresis (RNA, smaller DNA).
3. Pulsed-field gel electrophoresis (large DNA fragments).
What is the function of restriction endonucleases?
a. They splice short DNA pieces together.
b. They cleave DNA at specific sites.
c. They make RNA copies of DNA.
d. They make DNA copies from RNA.
Option b: They cleave DNA at specific sites.
Restriction Endonucleases (REs) functions:
1. Recognize specific DNA sequences (4-8 bp).
2. Cut DNA at precise locations.
3. Generate fragments with defined ends.
4. Enable DNA manipulation and cloning.
Types:
1. Type I: Cut randomly.
2. Type II: Cut specifically (e.g., EcoRI, HindIII).
3. Type III: Cut specifically, require ATP.
Applications:
1. Gene cloning
2. DNA sequencing
3. Genetic engineering
4. Forensic analysis
Options clarification
1. Option a: Ligases splice DNA.
2. Option c: Reverse transcriptase makes RNA copies.
3. Option d: Reverse transcriptase makes DNA from RNA.
Which of the following techniques uses RNA-guided enzymes?
a. Microarray
b. CRISPR
c. Immunohistochemistry
d. Restriction fragment mapping
Option b: CRISPR
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) utilizes RNA-guided enzymes:
Key Components:
1. Guide RNA (gRNA): Targets specific DNA sequence.
2. Cas enzyme (CRISPR-associated): Cuts DNA.
Applications:
1. Genome editing
2. Gene knockout/knockdown
3. Gene regulation
4. Gene therapy
Other options:
1. Option a: Microarray - measures gene expression.
2. Option c: Immunohistochemistry - detects proteins in tissues.
3. Option d: Restriction fragment mapping - analyzes DNA fragments.
To what does in situ hybridization refer?
a. Probes react with intact cells within tissues.
b. Probes are protected from degradation if hybridized.
c. RNA polymerase copies messenger RNA.
d. Hybridization takes place in solution.
Option a: Probes react with intact cells within tissues.
In situ hybridization (ISH) definition:
A laboratory technique detecting specific nucleic acid sequences (DNA/RNA) within intact cells/tissues, utilizing:
1. Labeled probes (DNA/RNA)
2. Hybridization
3. Visualization (fluorescence, chromogenic)
Applications:
1. Gene expression analysis
2. Localization of viral infections
3. Cancer research
4. Neurological disorders
Other options clarification:
1. Option b: RNA protection assay.
2. Option c: Reverse transcription.
3. Option d: Solution hybridization (e.g., Southern blot).
Which probe sequence will hybridize to 5′AGTCGATCGATGC3′?
a. 5′TCAGCTAGCTACG3′
b. 5′GCATCGATCGACT3′
c. 5′AGTCGATCGATGC3′
d. 5′CGTAGCTAGCTGA3′
Option b: 5′GCATCGATCGACT3′
This sequence is the reverse complement of the original sequence:
Original: 5′AGTCGATCGATGC3′
Reverse Complement: 5′GCATCGATCGACT3′
Base pairing rules:
A-T
G-C
Which best describes the principle of microarrays?
a. Arrays contain multiple unlabeled probes on a solid support.
b. Arrays contain multiple copies of one unique probe.
c. The sample is labeled with a fluorescent tag.
d. Hybridization is detected by the presence of radioactivity.
Option a: Arrays contain multiple unlabeled probes on a solid support.
Microarray Principle:
1. Thousands of unique, unlabeled DNA probes (spots) are immobilized on a solid surface (chip or slide).
2. Fluorescently labeled target samples (cDNA or RNA) hybridize to complementary probes.
3. Fluorescence intensity measures gene expression levels.
Key Characteristics:
1. High-throughput analysis
2. Parallel measurement of multiple genes
3. Quantitative gene expression profiling
Other options clarification:
Option b: Incorrect - multiple unique probes.
Option c: Partially correct - samples are labeled, but not the defining principle.
Option d: Incorrect - radioactivity detection is obsolete; fluorescence is standard.
Which best describes PCR?
a. Probes are joined by a ligating enzyme.
b. RNA copies of the original DNA are made.
c. Extender probes are used to detect a positive reaction.
d. Primers are used to make multiple DNA copies.
Option d: Primers are used to make multiple DNA copies.
Polymerase Chain Reaction (PCR) Process:
Key Steps
1. Denaturation: Double-stranded DNA unwinds.
2. Annealing: Primers bind complementary sequences.
3. Extension: DNA polymerase synthesizes new strands.
Essential Components
1. Primers (forward and reverse)
2. DNA template
3. DNA polymerase (e.g., Taq)
4. dNTPs (nucleotides)
5. Buffer
Applications
1. Gene amplification
2. Genetic testing
3. Forensic analysis
4. Gene cloning
Other options clarification:
Option a: Ligation (joining DNA fragments).
Option b: Reverse transcription (RNA from DNA).
Option c: Probe-based detection (e.g., real-time PCR).
During PCR, what happens in the annealing step?
a. The primers bind to the target DNA.
b. Strands are separated by heating.
c. An RNA copy is made.
d. Protein is made from the DNA strands.
Option a: The primers bind to the target DNA.
Annealing Step (PCR):
1. Cooling (typically 50-65°C) allows primer annealing.
2. Primers (forward and reverse) bind complementary target DNA sequences.
3. Specific base pairing (A-T, G-C) ensures precise binding.
PCR Cycle:
1. Denaturation (95-100°C): DNA unwinds.
2. Annealing (50-65°C): Primers bind.
3. Extension (72°C): DNA synthesis.
Other options clarification:
b. Denaturation (not annealing).
c. Reverse transcription (not PCR annealing).
d. Translation (protein synthesis, separate process).
What is the purpose of an amplification control in qPCR?
a. To avoid false positives
b. To ensure accuracy of target detection
c. To avoid contamination
d. To avoid false negatives
Option d: To avoid false negatives
Amplification controls in qPCR ensure that:
1. The reaction is working correctly.
2. Inhibitors are absent.
3. Equipment/functionality is validated.
By including controls, false negatives (incorrectly reporting absence of target) are minimized, ensuring reliable results.
Note: qPCR is less susceptible to amplicon contamination
Which technique is based on RNA amplification?
a. PCR
b. TMA
c. dPCR
d. SDA
Option b: TMA (Transcription-Mediated Amplification)
TMA is an RNA amplification technique:
Key Features
1. Utilizes RNA polymerase and reverse transcriptase.
2. Amplifies target RNA sequences.
3. Single-stranded RNA (ssRNA) or DNA (ssDNA) targets.
4. Isothermal reaction (constant temperature).
Other options:
a. PCR (Polymerase Chain Reaction): DNA amplification.
c. dPCR (Digital PCR): DNA quantification.
d. SDA (Strand Displacement Amplification): DNA amplification.
Which is used in Sanger sequencing?
a. UNG
b. ddNTP
c. ePCR
d. FRET
Option b: ddNTP (dideoxynucleotide triphosphates)
Sanger Sequencing (Chain Termination Method):
1. DNA template
2. Primer
3. DNA polymerase
4. ddNTPs (dideoxy ATP, CTP, GTP, TTP)
5. Normal NTPs
Process:
1. Extension: DNA synthesis.
2. Random incorporation: ddNTPs terminate strands.
3. Separation: Size-based gel electrophoresis.
4. Detection: Fluorescence or radiolabeling.
Other options:
a. UNG (Uracil-N-Glycosylase): PCR carryover prevention.
c. ePCR (emulsion PCR): Next-generation sequencing.
d. FRET (Fluorescence Resonance Energy Transfer): Real-time PCR, fluorescence detection.
What type of signal is generated in pyrosequencing?
a. Light
b. Fluorescence
c. Ionic conductance
d. Color
Option a: Light
Pyrosequencing:
1. DNA template
2. Primer
3. DNA polymerase
4. dNTPs (added sequentially)
Process:
1. Nucleotide incorporation releases pyrophosphate.
2. Pyrophosphate + ATP → ATP + light (via luciferase).
3. Light detected by photomultiplier.
Pyrosequencing advantages:
1. Real-time sequencing
2. High accuracy
3. Short read lengths (~300-500 bp)
Other options:
b. Fluorescence: Used in Sanger sequencing, qPCR.
c. Ionic conductance: Used in nanopore sequencing.
d. Color: Not directly related to pyrosequencing.
What is an NGS sequencing library?
a. A database of clinically significant sequences
b. A collection of software programs used for sequence analysis
c. A collection of short templates to be sequenced simultaneously
d. A list of all variants found in a sequencing run
Option c: A collection of short DNA/RNA templates prepared for simultaneous sequencing.
Next-Generation Sequencing (NGS) Library:
Key Components:
1. Fragmented DNA/RNA
2. Adapter ligation (indexing/barcoding)
3. Amplification (optional)
4. Quality control
Library Preparation:
1. Sample preparation
2. Library construction
3. Quality control
4. Sequencing
Types of NGS Libraries:
1. Whole-genome sequencing
2. Targeted sequencing
3. Transcriptome (RNA-seq)
4. Exome sequencing
Other options:
a. Database of clinically significant sequences (e.g., ClinVar).
b. Software programs (e.g., FASTQC, BWA).
d. List of variants (Variant Calling File, VCF).
In NGS, what is coverage?
a. The percentage of sequences carrying a variant
b. The number of genes sequenced
c. The number of times a region is sequenced
d. The percentage of the genome represented
Option c: The number of times a region is sequenced.
Coverage (Depth) in Next-Generation Sequencing (NGS):
- Average number of reads aligning to each genomic position.
- Measure of sequencing depth.
- Influences variant detection accuracy.
Types of Coverage:
1. Depth (average coverage)
2. Breadth (percentage of covered bases)
3. Uniformity (evenness of coverage)
Factors influencing coverage:
1. Sequencing technology
2. Library preparation
3. Genome complexity
4. Sequencing depth
Other options:
a. Allele frequency (percentage of sequences carrying a variant).
b. Gene content (number of genes sequenced).
d. Genome representation (percentage of genome covered).
