Molecular Diagnostic Techniques

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Questions and Answers

What is the term used to describe the true negative rate in diagnostic techniques?

Accuracy

Specificity (correct)

Reactivity

Sensitivity

Which of the following is NOT a benefit of molecular diagnosis?

Increased risk of contamination (correct)

Reduction in dependency on culture

Safe

High sensitivity

In electrophoresis, what determines the mobility of negatively charged molecules?

Shape and structure (correct)

Only charge

Only size

Size and charge

What is the purpose of hybridization in molecular diagnostic tests?

To detect specific DNA or RNA sequences Signup and view all the answers

Which of the following is an example of nuclear acid amplification in molecular diagnostic tests?

Target amplification Signup and view all the answers

What is the primary function of restriction enzymes in RFLP analysis?

To cut DNA at specific recognition nucleotide sequences Signup and view all the answers

What is the basis for analyzing differences among homologous DNA sequences using RFLP?

Sequence-specific recognition by restriction enzymes Signup and view all the answers

Why are restriction enzymes sequence-specific?

Because they recognize specific nucleotide sequences Signup and view all the answers

What is the outcome of RFLP analysis when two homologous DNA sequences have different restriction enzyme recognition sites?

The two sequences produce different RFLP patterns Signup and view all the answers

What is the importance of RFLP analysis in molecular diagnostics?

It enables the detection of differences among homologous DNA sequences Signup and view all the answers

What is the primary purpose of a probe in hybridization?

To detect complementary sequences in samples Signup and view all the answers

What characteristics of probes enable them to detect specific sequences?

Their high degree of specificity and various sizes Signup and view all the answers

What types of labels are commonly used to detect probes?

Radioisotopes, enzymes, or chemiluminescence Signup and view all the answers

What is the term for the binding of a probe to a complementary single-stranded sequence?

Hybridization Signup and view all the answers

What are some common applications of hybridization techniques?

Dot, in situ, Southern, Northern, and microarray Signup and view all the answers

What is the primary function of primers in PCR?

To bind to the region to be amplified Signup and view all the answers

What determines the product size in PCR?

The distance between the primer binding sites Signup and view all the answers

What is the purpose of reverse-transcriptase PCR?

To amplify RNA sequences Signup and view all the answers

What is a characteristic of isothermal amplification methods, such as LAMP?

They are highly sensitive and specific Signup and view all the answers

What is a potential limitation of PCR?

It is prone to false positives Signup and view all the answers

What is the primary purpose of the probe or dye in Real-Time PCR?

To generate a fluorescent signal from the product Signup and view all the answers

What is the characteristic of the signal obtained in Real-Time PCR?

An exponential curve with a lag phase, a log phase, and a stationary phase Signup and view all the answers

What is the relationship between the lag phase and the amount of starting material in Real-Time PCR?

The lag phase is inversely proportional to the amount of starting material Signup and view all the answers

What is the benefit of Real-Time PCR over traditional PCR?

Real-time quantification of the starting material Signup and view all the answers

What is the significance of the stationary phase in Real-Time PCR?

It is the phase where the reaction reaches a plateau Signup and view all the answers

Study Notes

Diagnostic Techniques

Diagnostic techniques used to identify pathogenic agents include: direct examination, culture, immunologic methods, and molecular analysis.

Molecular Diagnosis

Molecular diagnosis uses DNA, RNA, and proteins to identify pathogenic agents.
Advantages of molecular diagnosis include: • High sensitivity (true positive rate) • High specificity (true negative rate) • Reduced dependency on culture • Safe

Molecular Diagnostic Tests

Types of molecular diagnostic tests include: • Electrophoresis • Restriction fragment length polymorphism (RFLP) • Hybridization • Nuclear acid amplification (target) • Protein detection

Electrophoresis

Electrophoresis separates molecules in an electrophoretic field.
Negatively charged molecules move towards the positive end.
Mobility of molecules depends on: • Size: smaller molecules move faster • Structure: supercoiled molecules move faster than linear molecules, which move faster than nicked circles (in the case of DNA)

Restriction Fragment Length Polymorphism (RFLP)

RFLP involves analyzing differences among homologous DNA sequences through the use of restriction enzymes
Restriction enzymes cut DNA at specific recognition nucleotide sequences, which are sequence-specific
This technique is used to identify variations in DNA sequences by comparing the resulting fragment lengths

Hybridization

Hybridization involves binding of a probe to complementary single-stranded DNA sequences.
Denatured, single-stranded DNA is used in hybridization reactions.
Examples of hybridization techniques include Dot, in situ, Southern, Northern, and microarray.

Probe Characteristics

A probe is a fragment of nucleic acids used to detect complementary sequences in samples.
Probes can be labeled using radioisotopes, enzymes, or chemiluminescence.
Probes exhibit a high degree of specificity when binding to target sequences.
Probes can vary in size.

Nucleic Acid Amplification

Target amplification is an enzyme-mediated process that synthesizes copies of targeted nucleic acid
Two types of nucleic acid amplification are: Polymerase Chain Reaction (PCR) and Isothermal amplification (e.g. LAMP)

Polymerase Chain Reaction (PCR)

PCR is a highly sensitive method, but may produce false positives
PCR Primers are single-stranded DNA fragments complementary to sequences flanking the region to be amplified
The distance between the primer binding sites determines the size of the PCR product
Primers determine the specificity of the PCR reaction

Features of Primers

Types of primers: Random and Specific
Primer characteristics: • Product size • Annealing temperature • Specificity • Nucleotide composition

PCR Variations

Reverse-transcriptase PCR
Nested PCR
Multiplex PCR
Quantitative or real-time PCR

Real-Time or Quantitative PCR (qPCR)

qPCR uses a probe or dye to generate a fluorescent signal from the product.
The fluorescent signal is measured in real-time, allowing for quantification of the starting material.

qPCR Signal Curve

The qPCR signal curve is exponential in shape, consisting of three phases: lag phase, log phase, and stationary phase.
The lag phase is inversely proportional to the amount of starting material.

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Description

This quiz covers various molecular diagnostic techniques used to identify pathogenic agents, including DNA, RNA, and protein analysis. It also explores the advantages and types of molecular diagnostic tests.