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Chapter 5 : Introduction to Molecular Methodology

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Abstract:

This chapter provides a detailed discussion of the molecular techniques and equipment that are available, or under development, for use in the clinical laboratory. Molecular testing in the clinical laboratory consists of two major areas: (i) the use of DNA probes to directly detect or characterize a specific target and (ii) the use of nucleic acid amplification technologies to detect or characterize a specific target DNA or RNA. The use of DNA probe technology is discussed first in the chapter; nucleic acid amplification technology and nucleic acid sequencing are discussed later, followed by molecular arrays, a more elaborate application of probe technology. Each type of probe hybridization assay is discussed individually, as are probe amplification procedures. The primary objective of nucleic acid amplification techniques is to improve the sensitivity of assays based on nucleic acids and to eventually simplify these assays by development of automated assay formats such as real-time detection. Target amplification procedures probably provide the simplest products for post-amplification detection. In addition, dedicated procedures for the detection of amplified products following nucleic acid amplification, such as reverse dot blots, are discussed only briefly because these methods are being replaced with real-time nucleic acid amplification assays where the product is detected concurrently with the ongoing amplification. Real-time nucleic acid detection systems are also discussed in this chapter. Only through education and the ability to meet new challenges will clinical immunologists be able to control the use of molecular techniques and practice of molecular diagnostics in the clinical immunology laboratory.

Citation: Podzorski R. 2006. Introduction to Molecular Methodology, p 26-51. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch5

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Nucleic Acid Amplification Techniques
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Image of FIGURE 1
FIGURE 1

bDNA-based signal amplification. Target nucleic acid is released by cell disruption and captured to a solid surface via multiple contiguous capture extender probes. Label extender probes hybridize with adjacent target sequences and contain additional sequences homologous to the preamplifier probes. Preamplifier probes bind multiple bDNA (amplifier) probes. Enzyme-labeled oligonucleotides bind to the bDNA by homologous base pairing, and the enzyme-probe complex is measured by detection of chemiluminescence.

Citation: Podzorski R. 2006. Introduction to Molecular Methodology, p 26-51. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch5
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Image of FIGURE 2
FIGURE 2

Hybrid capture. Target DNA is released by cell disruption, denatured, and allowed to hybridize to specific RNA probes. Double-stranded DNA-RNA hybrids are captured to a solid surface via antibodies specific for the hybrids. Multiple reporter antibodies specific for the hybrids bind each captured DNA-RNA hybrid, setting the stage for signal amplification. A chemiluminescent substrate is added and the reporter antibody-hybrid complex is measured by detection of chemiluminescence.

Citation: Podzorski R. 2006. Introduction to Molecular Methodology, p 26-51. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch5
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Image of FIGURE 3
FIGURE 3

Invader. A primary probe, with a 5′ flap, and an invader probe bind to the target nucleic acid and form a 1-bp overlap. Cleavase recognizes this substrate and cleaves the 5′ flap from the primary probe. The free 5′ flap acts as secondary invader probe with the reporter probe. Cleavase cleaves the fluorescein (F) from the 5′ end of the reporter probe, separating it from the quencher dye (Q), allowing a fluorescent signal that can be detected. If the target region of the primary probe and the invader probe do not match perfectly with the target DNA, the proper substrate is not formed and the cleavase will not cleave the 5′ flap from the primary probe (right panel)

Citation: Podzorski R. 2006. Introduction to Molecular Methodology, p 26-51. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch5
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Image of FIGURE 4
FIGURE 4

PCR. (A) In the first cycle, a double-stranded DNA target sequence is used as template, with the primer binding sites indicated by the hatched lines. (B) The two DNA strands are separated by heat denaturation, and two synthetic oligonucleotide primers (complementary cross-hatched lines) anneal to their recognition sites in the 5′ to 3′ orientation when the reaction cools. Note that the 3′ ends of the primers (arrowheads) are facing each other. (C) DNA poly-merase initiates synthesis at the 3′ end of each primer. Extension of the primer via DNA polymerization (synthesis) results in new primer binding sites. The net result after one round of polymerization is one copy of each (two total) strand of the original target DNA of unspecified length. (D) In the second cycle, each of the four DNA strands shown in panel C anneals to primers (present in excess) to initiate a new round of DNA polymerization. Of the eight single-stranded products, two are of a length defined by the distance between and including the primer annealing site. This amplification product (amplicon) accumulates exponentially in subsequent cycles.

Citation: Podzorski R. 2006. Introduction to Molecular Methodology, p 26-51. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch5
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Image of FIGURE 5
FIGURE 5

TAS. The initial steps in the reaction involve formation of cDNAs from the target RNA by using oligonucleotide primers, one of which contains a T7 binding site. RNase H activity (a separate enzyme in NASBA, associated with the reverse transcriptase in TMA) degrades the initial strands of target RNA in the RNA-DNA hybrids after they have served as templates for the initial primer. The second primer then primes the initial single-stranded cDNAs, resulting in the formation of double-stranded cDNAs with one strand capable of serving as the transcription template for T7 RNA polymerase. This results in the synthesis of numerous copies of RNA. These RNAs serve as templates for synthesis of more cDNA intermediates. These cDNAs lead to the synthesis of more copies of RNA which then reenter the cycle.

Citation: Podzorski R. 2006. Introduction to Molecular Methodology, p 26-51. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch5
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Image of FIGURE 6
FIGURE 6

SDA. The initial rounds of the reaction (A) transform the original target sequence into the hemiphosphorothioate form with nickable BI sites at each end that enter into the second part of the reaction (B), which involves exponential amplification of the transformed target sequence. In reaction part A, sample DNA is denatured at 95°C in the presence of an excess of four specific primers that define the target sequence. Two primers, S1 and S2, contain unmodified BI recognition sites at their 5′ ends and specific target-binding sequences at their 3′ ends. S1 and S2 bind opposite strands of DNA flanking the target region. The other two primers, B1 and B2, are target-binding primers only, without a restriction endonuclease recognition sequence, and bind opposite strands of DNA immediately upstream of primers S1 and S2. The incorporation of primers B1 and B2 concomitantly generates a product with defined ends during the reaction and eliminates the need for restriction enzyme cleavage of the sample DNA prior to SDA. Following the addition of the primers, the reaction mixture is allowed to cool to 37°C, and DNA polymerase and BI are added in excess together with dATP, dGTP, dTTP, and dCTPαS. The DNA polymerase activity now extends all four primers simultaneously (A). Primers S1 and S2 form complementary strands of modified DNA that contain unmodified BI sites at their 5′ ends. B1 and B2 prime the same strands and displace the newly synthesized strands primed with S1 and S2, producing new strands of DNA with defined ends that start immediately upstream of the S1 and S2 binding sites. Now S1 and B1 bind to the displaced strand initially primed with S2, while S2 and B2 bind to the displaced strand initially primed with S1 (A). Extension and displacement reactions on these templates produce two defined fragments with a hemiphosphorothioate BI site at each end. Copies of the original target DNA containing hemiphosphorothioate BI ends have now been generated (A, bottom). These copies now enter the second, and prominent, part of the SDA reaction (B). Following priming and extension from S1 and S2, a double-stranded fragment of a specific size that contains a BI site on each end that is susceptible to nicking (remember that S1 and S2 primers contain unmodified BI recognition sites at their 5′ ends) is generated. Repeated cycles of nicking, DNA polymerization and strand displacement, and priming of the displaced strands with S1 and S2 result in exponential amplification of target DNA. For product detection, the fluorogenic probe (large circle) binds to one strand of amplified DNA and its 3′ end is extended simultaneously with the amplification (S1) probe for that strand. The extended fluorogenic probe is displaced by the product from the amplification (S1) probe. The extended fluorogenic probe is now bound by the opposite-strand primer (S2) and is copied. The copying of the fluorogenic probe forces the stem-loop structure apart and creates a double-stranded BI site, which is flanked by both FAM (small open circle) and ROX (small solid circle). The BI site in the fluorogenic probe lacks the dCTPαS at the nucleotide position of BI cleavage. As a result, BI cuts clean through the two strands of DNA and liberates FAM from the quencher. Now fluorescent emission from FAM can be detected.

Citation: Podzorski R. 2006. Introduction to Molecular Methodology, p 26-51. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch5
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Image of FIGURE 7
FIGURE 7

TaqMan. Annealing of the reporter probe to one specific strand of the PCR product during the course of amplification generates a target-specific substrate suitable for exonuclease cleavage. During DNA extension from a PCR primer, the 5′ → 3′ exonuclease activity of cleaves 5′-terminal nucleotides off the bound reporter probe and frees the FAM reporter (F) from the TAMRA quencher (Q). The free FAM reporter now emits fluorescence.

Citation: Podzorski R. 2006. Introduction to Molecular Methodology, p 26-51. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch5
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Image of FIGURE 8
FIGURE 8

Molecular beacons. During the denaturation step of PCR, the target DNA and the stem-loop of the molecular beacon denature. As the temperature is lowered to allow annealing of PCR primers, the molecular beacon hybridizes to one specific strand of the PCR product. This conforma-tional change that occurs during hybridization forces the stem apart and causes the fluorescent dye (F) to move away from the quencher (Q), leading to fluorescence. When the temperature is raised for primer extension, the molecular beacons dissociate from their targets and do not interfere with PCR.

Citation: Podzorski R. 2006. Introduction to Molecular Methodology, p 26-51. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch5
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Image of FIGURE 9
FIGURE 9

Hybridization probes. Two separate fluorescently labeled probes (the donor probe is labeled at the 3′ end with fluorescein [probe D] and the acceptor probe is labeled at the 5′ end with LightCycler Red 640 [probe A]) are juxtaposed tail to head upon specifically binding to one strand of the PCR product during the annealing phase of PCR. An excitation wavelength of light specific for the donor probe only is produced during annealing. The acceptor probe absorbs resonance energy from the donor probe and emits fluorescence, with emission collection only being done in the acceptor range.

Citation: Podzorski R. 2006. Introduction to Molecular Methodology, p 26-51. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch5
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Image of FIGURE 10
FIGURE 10

Basic components of a real-time nucleic acid amplification instrument. A computer and associated software run the instrument and analyze the amplification data. A thermal cycler provides the cycling temperature conditions for nucleic acid amplification. The optical system includes components to excite the fluorescent reporter molecules, together with an emission detector.

Citation: Podzorski R. 2006. Introduction to Molecular Methodology, p 26-51. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch5
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Image of FIGURE 11
FIGURE 11

Bases used in Sanger’s dideoxy chain termination procedure.

Citation: Podzorski R. 2006. Introduction to Molecular Methodology, p 26-51. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch5
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Image of FIGURE 12
FIGURE 12

Cycle sequencing-based procedure. DNA or RNA to be sequenced is extracted from the sample of interest, and PCR is used to amplify the target to be sequenced. Once the target has been cleaned up it is used as template in a dideoxy-based cycle sequencing reaction. An automated sequencer determines the composition of the sequence template based upon different-colored fluorescent chain terminators and directly enters the sequence data into a computerized workstation, where data analysis is completed.

Citation: Podzorski R. 2006. Introduction to Molecular Methodology, p 26-51. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch5
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Image of FIGURE 13
FIGURE 13

Expression array schema. Templates for genes of interest are obtained from DNA clones and amplified by PCR. Following purification, the aliquots (≈5 nl) are “printed” on glass microscope slides using a high-speed computer-controlled robot that draws the capture probes from a microtiter plate (left side of figure). Total RNA synthesized from test cDNA (stimulated T cells, for example) and control cDNA (unstimulated T cells) is labeled with different-colored fluorescent dyes during a single round of reverse transcription (right side of figure). The labeled test RNAs are pooled and allowed to hybridize under stringent conditions to the capture probes on the microarray. Laser excitation of the fluorescent dyes yields an emission of known spectra, which is measured using a scanning confocal laser microscope. Data from a single hybridization experiment are viewed as a normalized ratio comparing the intensity of the signal between the two dyes. Significant deviations from background are indicative of increased or decreased levels of gene expression relative to the control sample.

Citation: Podzorski R. 2006. Introduction to Molecular Methodology, p 26-51. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch5
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Image of FIGURE 14
FIGURE 14

Photolithography. (A) A 1.2- by 1.2-cm glass substrate with photoprotected linker groups. Areas of the glass substrate are selectively illuminated by light passing through a photolithographic mask. Deprotected areas are activated. (B) With nucleoside incubation, chemical coupling occurs at activated positions. This process is repeated until the desired set of probes is obtained. This type of microarray is called a GeneChip. The microarray is placed into a cartridge to facilitate its use in a hybridization assay.

Citation: Podzorski R. 2006. Introduction to Molecular Methodology, p 26-51. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch5
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Image of FIGURE 15
FIGURE 15

Protocol for using the GeneChip. Target DNA or RNA is amplified by PCR using primers with a T3 RNA polymerase promoter sequence in one and a T7 RNA polymerase promoter sequence in the other. The PCR product is transcribed (DNA → RNA) using T7 or T3 RNA polymerase in the presence of fluorescein-labeled rUTP The fluorescein-labeled RNA is fragmented by heating (95°C for 30 min) in the presence of 30 mM MgCl. The labeled and fragmented RNA is hybridized to the GeneChip and then analyzed by laser scanning and computer analysis of the resulting fluorescence.

Citation: Podzorski R. 2006. Introduction to Molecular Methodology, p 26-51. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch5
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Tables

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TABLE 1

Comparison of basic features of five real-time PCR instruments

Citation: Podzorski R. 2006. Introduction to Molecular Methodology, p 26-51. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch5

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