Types of PCR: Multi-plex PCR

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Questions and Answers

What is the purpose of multiplex PCR?

To amplify multiple products simultaneously (correct)

To measure gene expression

To amplify a single product

To convert RNA into DNA

What is the first step in reverse transcription polymerase chain reaction (RT-PCR)?

Conversion of RNA into cDNA using reverse transcriptase

MRNA can be amplified directly by PCR.

False

Which of the following is NOT a step in RT-PCR?

Amplify target RNA directly Signup and view all the answers

What is the role of the oligo(T) fragment in RT-PCR?

To bind to the 3’ poly(A) tail of each mRNA Signup and view all the answers

What does real-time PCR measure?

The amount of PCR product at the end of each cycle Signup and view all the answers

The threshold cycle, or ______, is the PCR cycle at which sample fluorescence crosses the threshold.

CT Signup and view all the answers

What type of fluorescent dye can be used in real-time PCR?

SYBR Green Signup and view all the answers

Nested PCR increases sensitivity in clinical applications.

True Signup and view all the answers

What is positional cloning?

Positional cloning is a laboratory approach used to locate the position of a disease-associated gene on a chromosome. Signup and view all the answers

Who invented the RFLP technique?

Alec Jeffreys Signup and view all the answers

RFLP markers are typically dominant in genetic analysis.

False Signup and view all the answers

The sickle-cell mutation changes an A–T base pair to a _____ base pair.

T–A Signup and view all the answers

What disease is associated with the mutation in the gene for β-globin?

Sickle-cell anemia Signup and view all the answers

What is genetic polymorphism?

Inherited genetic differences found in over 1% of a normal population Signup and view all the answers

What is the role of RFLP probes?

RFLP probes are labeled DNA sequences that hybridize with DNA fragments to reveal unique blotting patterns. Signup and view all the answers

Sickle-cell anemia results in the abnormal form of hemoglobin called _____

Hb-S Signup and view all the answers

Study Notes

Multi-plex PCR

More than one primer pair can be included in a single PCR reaction.
Enables simultaneous amplification of multiple DNA products.
Particularly useful for typing or identification analyses.
Requires complex optimization of reagents and conditions.
Target sequences may amplify at different efficiencies, posing challenges.
Potential primer interference can affect binding to target sequences.
PCR conditions must be carefully adjusted to ensure optimal amplification.

Reverse Transcriptase PCR (RT-PCR)

Measures gene expression by converting RNA to cDNA before amplification.
cDNA synthesis is catalyzed by reverse transcriptase, utilizing mRNA as a template.
mRNA is fragile and cannot be amplified directly by PCR.
Reverse transcriptase was first discovered in retroviruses, which have RNA genomes.
The RT-PCR process includes:
- Binding an oligo(T) primer to the 3’ poly(A) tail of mRNA.
- Synthesizing cDNA strands from RNA templates.
- Separating RNA-cDNA hybrids through temperature increase.
- Using gene-specific primers for further amplification.
Final amplification results in billions of copies of the expressed gene.
The quantity of DNA is visualized using gel electrophoresis.

Nested PCR

Enhances sensitivity essential in clinical applications where specimen quality is often compromised.
Addresses challenges posed by low target abundance and interfering sequences.
Increases reliability of PCR outcomes in clinical diagnostics.

Real-Time (Quantitative) PCR (qPCR)

Measures PCR product amounts at each amplification cycle in real-time.
Uses fluorescent dyes for detection during or after the amplification process.
Examples include SYBR Green and ethidium bromide (EtBr) for dye-based detection.
Detection methods:
- DNA intercalating or minor groove-binding dyes that fluoresce more as template DNA increases.
- Sequence-specific DNA probes labeled with fluorophores that rely on Taq polymerase’s exonuclease activity.
The polymerase separates fluorophores from quencher molecules, leading to increased fluorescence.
The threshold cycle (CT) indicates the PCR cycle number at which fluorescence crosses a pre-defined threshold.
Fluorescence accumulation is represented as a sigmoid curve over multiple cycles, with earlier fluorescence indicating more starting template.

Positional Cloning

Laboratory technique used to locate disease-associated genes on chromosomes.
Functions without prior knowledge of the gene's protein role in the disease.
Utilizes known polymorphic markers to trace inheritance patterns in affected families.

Steps in Positional Cloning

Begins with genetic analysis of patients and their families to define critical DNA regions.
Statistical estimation determines if disease traits segregate independently or are linked to specific molecular markers.
Identification of markers linked to disease manifestations.

Restriction Fragment Length Polymorphism (RFLP)

Technique developed by Alec Jeffreys in 1984 for studying hereditary diseases.
Analyzes unique patterns in DNA fragments through Variable Number of Tandem Repeats (VNTRs).
Genetic polymorphism refers to inherited genetic differences in over 1% of the population.
RFLP exploits DNA sequence differences for genetic differentiation (intraspecies and interspecies).
Markers are typically co-dominant and highly locus-specific, allowing detection of both alleles in heterozygous samples.

RFLP Probes

Labeled DNA sequences that hybridize with digested DNA fragments separated via gel electrophoresis.
Reveal unique blotting patterns for specific genotypes at designated loci.
Used for genome mapping, genetic variation analysis, forensics, paternity testing, and hereditary disease diagnosis.

Application: Sickle Cell Anemia Detection by RFLP

A mutation causes the alteration of restriction sites within the gene or flanking regions.
Sickle-cell anemia involves a single base-pair change resulting in Hb-S instead of normal Hb-A.
The mutation modifies the sixth codon of β-globin from GAG (glutamic acid) to GUG (valine), affecting hemoglobin structure.
Abnormal aggregation of Hb-S leads to the characteristic sickling of red blood cells.

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Description

This quiz covers the concept of Multiplex PCR, which allows the amplification of multiple DNA sequences simultaneously. Learn about the importance of primer pairs, optimization of reagents, and the challenges involved in ensuring efficient amplification. Test your knowledge on this advanced PCR technique.