7 Molecular Diagnostics Lec II

Molecular Diagnostic Tools
Nucleic acid Based diagnostic tools and Approaches to Disease Diagnosis (Lec II)
Dr. Anum Gul

(BT-502)
Polymerase Chain Reaction (PCR) in Diagnostics
🠶 PCR is a molecular technique to amplify a single copy of DNA and
generates millions of copies of a particular DNA sequence derived from a
patient’s sample (blood and tissue).
🠶 This method relies on thermal cycling of repeated heating and cooling of the
reaction for DNA, causing replication of the DNA.

Quantitative PCR (qPCR) in Diagnostics
🠶 In qPCR, the double-stranded DNA product is bound by a fluorescent dye
and the fluorescence is measured after each amplification cycle to quantify
the amount of product in real time.
🠶 Post-PCR processing is not required, and therefore, time to pathogen
identification is shorter than for conventional PCR.

🠶 DNA primers are required for the initiation of DNA synthesis.
🠶 The vast majority of PCR methods use thermal cycling, as these are necessary
to separate two DNA strands of a double helix at a high temperature; this
process is called DNA melting.
🠶 The number of copies of a target DNA in a clinical sample can be determined

by obtaining the CT value for the sample and extrapolating the starting
amount from the standard curve.
Real-time PCR may be described as occurring in four phases.
🠶 In the first, or linear, phase (generally about 10 to 15 cycles), fluorescence

emission at each cycle has not yet risen above the background level.
🠶 In the early exponential phase, a sufficient amount of double-stranded DNA

has been produced to increase the amount of fluorescence above a threshold
level that is significantly higher than the background. The cycle at which this
occurs is known as the threshold cycle (CT).
🠶 The CT value is inversely correlated with the amount of target DNA in the
original sample.
🠶 During the exponential phase, the amount of fluorescence doubles as the

DNA products of the reaction double in each cycle under ideal conditions.
🠶 In the plateau phase, the reaction components become limited and

measurements of the fluorescence intensity are no longer useful.
🠶 When a pathogen cannot be detected using available primers, broad-range PCR

may be used.
🠶 This approach employs universal primers that anneal to a common nucleotide

sequence, for example, the 16S rRNA gene present in all bacteria.
🠶 Following amplification, products are sequenced for pathogen identification by

comparison of the sequence to a database.
🠶 Additionally, a variant of real-time PCR may be used to quantify the levels

of a variety of mRNAs in different eukaryotic tissues or prokaryotic cells.
🠶 In these instances, the initial target is RNA and not DNA, and therefore, a
reverse transcription (RT) step is needed to generate cDNA before the real-
time PCR.
🠶 With quantitative RT-PCR, it is possible to measure mRNA levels that are

about 10,000- to 100,000-fold lower than those measurable by traditional
techniques.
Application of PCR technology
Medical research and clinical medicine are benefiting from PCR technology
mainly in two areas:
(i) Detection of infectious organisms,
(ii) Detection of genetic variations, including mutations, in human genes.

Infectious disease
🠶 It is used to detect the DNA or RNA of pathogenic organisms such as viruses and
bacteria.
🠶 To detect the presence of pathogenic agents earlier than serologically based methods,
as patients can take weeks to develop antibodies against an infectious agent.
🠶 It can reveal specific characteristics of that pathogen, for example, the presence of
antibiotic resistance genes that can be used to determine the best course of treatment.
Blood screening
⮚ Serological tests have been used to screen donated blood samples for the presence
of infectious agents.
⮚ Nevertheless, a small risk of viral transmission remains primarily due to the failure
of such screening tests to identify recently infected donors during the period (the
time delay post-infection in which the body develops an immune response to the
infectious agent).
🠶 Moreover, tests using PCR nucleic acid amplification testing (NAT)

technology detect the actual virus.
🠶 Experts consider that including such tests in blood screening programs

could provide an additional measure of protection by detecting viral
infection at an earlier stage.
Genetic testing
We all know that PCR technology can be used to easily distinguish among
the tiny variations in DNA that make people genetically unique.
In the future, PCR technology may be used in predictive test methods for
finding out who is predisposed to common disorders, such as heart disease
and many cancers.
Detection of Multiple Disease-Associated
Mutations Using Microarrays
🠶 A DNA microarray (DNA chip or gene chip) experiment consists of

hybridizing a nucleic acid sample (target) derived from the mRNAs of a cell
or tissue to probes that are arrayed on a solid platform.
🠶 Depending on the purpose of the experiment, the probes on a microarray

may represent an entire genome, a single chromosome, selected genomic
regions, or selected coding regions from one or several different organisms.
🠶 Some DNA microarrays contain sets of oligonucleotides as probes, usually
representing thousands of genes that are synthesized directly on a solid
surface.
🠶 The probes are typically 20 to 70 nucleotides, although longer probes can

also be used, and several probes with different sequences for each gene are
usually present on the microarray to minimize errors.
🠶 Probes are designed to be specific for their target sequences, to avoid

hybridization with nontarget sequences, and to have similar melting
(annealing) temperatures so that all target sequences can bind to their
complementary probe sequence under the same conditions.
🠶 A complete whole-genome oligonucleotide array may contain more than

500,000 probes representing as many as 30,000 genes.
🠶 Methods to calibrate the data among microarrays in an experiment include using

the fluorescence intensity of a gene that is not differentially expressed among
different conditions as a reference point (i.e., a housekeeping gene).
🠶 Genes whose expression changes in response to a particular biological condition

are identified by comparing the fluorescence intensities for each gene, averaged
among replicates, under two different conditions.
🠶 The raw data of the fluorescence emissions of each gene are converted to a ratio,
commonly expressed as fold change.
🠶 Generally, positive ratios represent greater expression of the gene in the test
sample than in the reference sample.
🠶 Negative values indicate a lower level of expression in the test sample

relative to the reference sample.
Detection of Epigenetic Markers
🠶 Epigenetic modifications refer to the covalent modification of DNA or

histones associated with DNA.
🠶 Specific bases in DNA may be methylated, and acetyl, ubiquitin, methyl,

phosphate groups may be added to specific amino acids in histone
proteins.
🠶 Epigenetic changes alter gene expression and cellular phenotype, although

they do not change the genotype of a cell.
DNA methylation almost always involves the addition of a methyl group to a

cytosine residue in CpG dinucleotides, generating 5-methylcytosine.
🠶 The addition is catalyzed by a DNA methyltransferase.
🠶 About 75% of the 28 million CpG dinucleotides in the mammalian genome are
methylated.
🠶 DNA methylation is a normal process that serves a regulatory function.
🠶 It downregulates gene expression in response to environmental conditions by

inhibiting binding of transcription factors or by recruiting specific methyl-
binding domain proteins that induce the formation of heterochromatin.
🠶 Recently, altered DNA methylation patterns have been found to be

associated with a number of human diseases, especially cancers.
🠶 In tumors, levels of genome methylation generally decrease; however, the

promoters of some genes exhibit increased methylation of normally
unmethylated CpG dinucleotides.
🠶 Methylation of core promoter sequences inactivates expression of these

genes, some of which encode tumor suppressors.
🠶 Thus, epigenetic modification can have an effect similar to that of a

mutation or deletion.
🠶 However, because DNA methylation is an epigenetic alteration, it is, at

least in theory, reversible and therefore may be a target for demethylation
drugs to treat human disease.
Strategies to detect methylated sequences
(i) methylation-sensitive restriction endonucleases that bind to DNA only when CpG
dinucleotides in their recognition site are methylated (or only when they are
unmethylated).
(ii) 5-methylcytosine-specific antibodies to capture methylated fragments that can

be identified using a microarray or by DNA sequencing.
(iii) Chemical deamination of nonmethylated

cytosines to uracils using sodium bisulfite;
methylated cytosines are protected against
deamination.
The last method converts differences in methylation

patterns into differences in sequence (i.e., C to U)
that can be identified by PCR, sequencing, or
microarrays.
Detection of SNPs by Mass Spectrometry
🠶 Mass spectrometry is an analytical technique in which samples are ionized

into charged molecules and ratio of their mass-to-charge (m/z) can be
measured.
🠶 In MALDI-TOF mass spectrometry, the ion source is matrix-assisted laser

desorption/ionization (MALDI), and the mass analyzer is time-of-flight
(TOF) analyzer.
MALDI methodology is a three-step process.
🠶 First, the sample is mixed with a suitable matrix material and applied to a metal
plate.
🠶 Second, a pulsed laser irradiates the sample, triggering ablation and desorption
of the sample and matrix material.
🠶 Finally, the analyte molecules are ionized by being protonated or deprotonated

in the hot plume of ablated gases and are separated in an electromagnetic field
and identified by their mass-to-charge ratio.
Detection of SNPs by MALDI-TOF
Advantages of MALDI-TOF
🠶 High accuracy and sensitivity
🠶 MALDI is a soft ionization method without fragmenting or decomposing the analyte.
🠶 MALDI is appropriate to analyze biomolecules like peptides, lipids, saccharides, or

other organic macromolecules.
🠶 Small amount of sample is needed
🠶 No fluorescent labelling is needed
🠶 Large number of SNPs can be detected in single reaction

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Molecular Diagnostic Tools

Dr. Anum Gul

🠶 PCR is a molecular technique to amplify a single copy of DNA and

generates millions of copies of a particular DNA sequence derived from a

patient’s sample (blood and tissue).

reaction for DNA, causing replication of the DNA.

🠶 In qPCR, the double-stranded DNA product is bound by a fluorescent dye

and the fluorescence is measured after each amplification cycle to quantify

the amount of product in real time.

🠶 Post-PCR processing is not required, and therefore, time to pathogen

identification is shorter than for conventional PCR.

🠶 DNA primers are required for the initiation of DNA synthesis.

🠶 The number of copies of a target DNA in a clinical sample can be determined

Real-time PCR may be described as occurring in four phases.

🠶 In the first, or linear, phase (generally about 10 to 15 cycles), fluorescence

🠶 In the early exponential phase, a sufficient amount of double-stranded DNA

🠶 During the exponential phase, the amount of fluorescence doubles as the

🠶 In the plateau phase, the reaction components become limited and

🠶 When a pathogen cannot be detected using available primers, broad-range PCR

🠶 This approach employs universal primers that anneal to a common nucleotide

🠶 Following amplification, products are sequenced for pathogen identification by

🠶 Additionally, a variant of real-time PCR may be used to quantify the levels

🠶 With quantitative RT-PCR, it is possible to measure mRNA levels that are

(i) Detection of infectious organisms,

(ii) Detection of genetic variations, including mutations, in human genes.

🠶 Moreover, tests using PCR nucleic acid amplification testing (NAT)

🠶 Experts consider that including such tests in blood screening programs

🠶 A DNA microarray (DNA chip or gene chip) experiment consists of

🠶 Depending on the purpose of the experiment, the probes on a microarray

🠶 The probes are typically 20 to 70 nucleotides, although longer probes can

🠶 Probes are designed to be specific for their target sequences, to avoid

🠶 A complete whole-genome oligonucleotide array may contain more than

🠶 Methods to calibrate the data among microarrays in an experiment include using

🠶 Genes whose expression changes in response to a particular biological condition

🠶 Negative values indicate a lower level of expression in the test sample

🠶 Epigenetic modifications refer to the covalent modification of DNA or

🠶 Specific bases in DNA may be methylated, and acetyl, ubiquitin, methyl,

🠶 Epigenetic changes alter gene expression and cellular phenotype, although

DNA methylation almost always involves the addition of a methyl group to a

🠶 The addition is catalyzed by a DNA methyltransferase.

🠶 DNA methylation is a normal process that serves a regulatory function.

🠶 It downregulates gene expression in response to environmental conditions by

🠶 Recently, altered DNA methylation patterns have been found to be

🠶 In tumors, levels of genome methylation generally decrease; however, the

🠶 Methylation of core promoter sequences inactivates expression of these

🠶 Thus, epigenetic modification can have an effect similar to that of a

🠶 However, because DNA methylation is an epigenetic alteration, it is, at

(ii) 5-methylcytosine-specific antibodies to capture methylated fragments that can

(iii) Chemical deamination of nonmethylated

The last method converts differences in methylation

🠶 Mass spectrometry is an analytical technique in which samples are ionized

🠶 In MALDI-TOF mass spectrometry, the ion source is matrix-assisted laser

MALDI methodology is a three-step process.

🠶 Finally, the analyte molecules are ionized by being protonated or deprotonated

🠶 High accuracy and sensitivity

🠶 MALDI is a soft ionization method without fragmenting or decomposing the analyte.

🠶 MALDI is appropriate to analyze biomolecules like peptides, lipids, saccharides, or

🠶 Small amount of sample is needed

🠶 No fluorescent labelling is needed

🠶 Large number of SNPs can be detected in single reaction