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Chapter 5 : Introduction to Molecular Methodology
Author:
Raymond P. Podzorski
Content Type: Reference
Publication Year:
2006
Category: Immunology
Book DOI:
10.1128/9781555815905
Chapter DOI:
10.1128/9781555815905.ch5
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Introduction to Molecular Methodology, Page 1 of 2
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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
Figures
Introduction to Molecular Methodology
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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
/content/10.1128/9781555815905.ch05.ch05fig01
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.
10.1128/9781555815905/f0028-01_thmb.gif
10.1128/9781555815905/f0028-01.gif
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
/content/10.1128/9781555815905.ch05.ch05fig02
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.
10.1128/9781555815905/f0029-01_thmb.gif
10.1128/9781555815905/f0029-01.gif
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
/content/10.1128/9781555815905.ch05.ch05fig03
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)
10.1128/9781555815905/f0030-01_thmb.gif
10.1128/9781555815905/f0030-01.gif
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
/content/10.1128/9781555815905.ch05.ch05fig04
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) Taq 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.
10.1128/9781555815905/f0032-01_thmb.gif
10.1128/9781555815905/f0032-01.gif
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) Taq 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
/content/10.1128/9781555815905.ch05.ch05fig05
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.
10.1128/9781555815905/f0033-01_thmb.gif
10.1128/9781555815905/f0033-01.gif
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
/content/10.1128/9781555815905.ch05.ch05fig06
FIGURE 6
SDA. The initial rounds of the reaction (A) transform the original target sequence into the hemiphosphorothioate form with nickable BsoBI 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 BsoBI 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 Bst DNA polymerase and BsoBI are added in excess together with dATP, dGTP, dTTP, and dCTPαS. The Bst DNA polymerase activity now extends all four primers simultaneously (A). Primers S1 and S2 form complementary strands of modified DNA that contain unmodified BsoBI 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 BsoBI site at each end. Copies of the original target DNA containing hemiphosphorothioate BsoBI 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 BsoBI site on each end that is susceptible to nicking (remember that S1 and S2 primers contain unmodified BsoBI recognition sites at their 5′ ends) is generated. Repeated cycles of nicking, Bst 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 BsoBI site, which is flanked by both FAM (small open circle) and ROX (small solid circle). The BsoBI site in the fluorogenic probe lacks the dCTPαS at the nucleotide position of BsoBI cleavage. As a result, BsoBI cuts clean through the two strands of DNA and liberates FAM from the quencher. Now fluorescent emission from FAM can be detected.
10.1128/9781555815905/f0035-01_thmb.gif
10.1128/9781555815905/f0035-01.gif
FIGURE 6
SDA. The initial rounds of the reaction (A) transform the original target sequence into the hemiphosphorothioate form with nickable BsoBI 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 BsoBI 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 Bst DNA polymerase and BsoBI are added in excess together with dATP, dGTP, dTTP, and dCTPαS. The Bst DNA polymerase activity now extends all four primers simultaneously (A). Primers S1 and S2 form complementary strands of modified DNA that contain unmodified BsoBI 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 BsoBI site at each end. Copies of the original target DNA containing hemiphosphorothioate BsoBI 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 BsoBI site on each end that is susceptible to nicking (remember that S1 and S2 primers contain unmodified BsoBI recognition sites at their 5′ ends) is generated. Repeated cycles of nicking, Bst 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 BsoBI site, which is flanked by both FAM (small open circle) and ROX (small solid circle). The BsoBI site in the fluorogenic probe lacks the dCTPαS at the nucleotide position of BsoBI cleavage. As a result, BsoBI 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
/content/10.1128/9781555815905.ch05.ch05fig07
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 Taq 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.
10.1128/9781555815905/f0037-01_thmb.gif
10.1128/9781555815905/f0037-01.gif
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 Taq 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
/content/10.1128/9781555815905.ch05.ch05fig08
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.
10.1128/9781555815905/f0037-02_thmb.gif
10.1128/9781555815905/f0037-02.gif
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
/content/10.1128/9781555815905.ch05.ch05fig09
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.
10.1128/9781555815905/f0039-01_thmb.gif
10.1128/9781555815905/f0039-01.gif
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
/content/10.1128/9781555815905.ch05.ch05fig10
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.
10.1128/9781555815905/f0041-01_thmb.gif
10.1128/9781555815905/f0041-01.gif
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
/content/10.1128/9781555815905.ch05.ch05fig11
FIGURE 11
Bases used in Sanger’s dideoxy chain termination procedure.
10.1128/9781555815905/f0042-01_thmb.gif
10.1128/9781555815905/f0042-01.gif
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
/content/10.1128/9781555815905.ch05.ch05fig12
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.
10.1128/9781555815905/f0042-02_thmb.gif
10.1128/9781555815905/f0042-02.gif
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
/content/10.1128/9781555815905.ch05.ch05fig13
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.
10.1128/9781555815905/f0044-01_thmb.gif
10.1128/9781555815905/f0044-01.gif
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
/content/10.1128/9781555815905.ch05.ch05fig14
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.
10.1128/9781555815905/f0045-01_thmb.gif
10.1128/9781555815905/f0045-01.gif
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
/content/10.1128/9781555815905.ch05.ch05fig15
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 MgCl2. The labeled and fragmented RNA is hybridized to the GeneChip and then analyzed by laser scanning and computer analysis of the resulting fluorescence.
10.1128/9781555815905/f0046-01_thmb.gif
10.1128/9781555815905/f0046-01.gif
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 MgCl2. 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
References
/content/book/10.1128/9781555815905.ch05
1.
Albert, T. J.,, J. Norton,, M. Ott,, T. Richmond,, K. Nuwaysir,, E. F. Nuwaysir,, K.-P. Stengele, and , R. D. Green. 2003. Light-directed 5′→3′ synthesis of complex oligonucleotide microarrays. Nucleic Acids Res. 31: e35.
2.
Arnheim, N.,, T. White, and , W. E. Rainey. 1990. Application of PCR: organismal and population biology. BioScience 40: 174– 182.
3.
Arnold, L. J., Jr.,, P. W. Hammond,, W. A. Wiese, and , N. C. Nelson. 1989. Assay formats involving acridinium-ester-labeled DNA probes. Clin. Chem. 35: 1588– 1594.
4.
Aspinall, S.,, A. D. Steele,, I. Peenze, and , M. J. Mphahlele. 1995. Detection and quantitation of hepatitis B virus DNA: comparison of two commercial hybridization assays with polymerase chain reaction. J. Viral Hepat. 2: 107– 111.
5.
Bieche, I.,, P. Onody,, I. Laurendeau,, M. Olivi,, D. Vidaud,, R. Lidereau, and , M. Vidaud. 1999. Real-time reverse transcription-PCR assay for future management of ERBB2-based clinical applications. Clin. Chem. 45: 1148– 1156.
6.
Bonnet, G.,, S. Tyagi,, A. Libchaber, and , F. R. Kramer. 1999. Thermodynamic basis of the enhanced specificity of structured DNA probes. Proc. Natl. Acad. Sci. USA 96: 6171– 6176.
7.
Britten, R. J., and , E. H. Davidson. 1985. Hybridization strategy, p. 3– 14. In B. D. Hames and , S. J. Higgins (ed.), Nucleic Acid Hybridization: a Practical Approach. IRL Press, Oxford, United Kingdom.
8.
Brown, T. 1993. Southern blotting, p. 2.9.1– 2.9.15. In F. M. Ausubel, , R. Brent,, R. E. Kingston,, D. D. Moore,, J. G. Seidman, and , K. Struhl (ed.), Current Protocols in Molecular Biology, vol. 1. John Wiley & Sons, New York, N.Y.
9.
Brown, T. 1993. Dot and slot blotting of DNA, p. 2.9.15– 2.9.20. In F. M. Ausubel,, R. Brent,, R. E. Kingston,, D. D. Moore,, J. G. Seidman, and , K. Struhl (ed.), Current Protocols in Molecular Biology, vol. 1. John Wiley & Sons, New York, N. Y.
10.
Brown, T. 1993. Analysis of RNA by Northern and slot blot hybridization, p. 4.9.1– 4.9.14. In F. M. Ausubel,, R. Brent,, R. E. Kingston,, D. D. Moore,, J. G. Seidman, and , K. Struhl (ed.), Current Protocols in Molecular Biology, vol. 1. John Wiley & Sons, New York, N.Y.
11.
Castle, P. E.,, A. T. Lorincz,, D. R. Scott,, M. E. Sherman,, A. G. Glass,, B. B. Rush,, S. Wacholder,, R. D. Burk,, M. M. Manos,, J. E. Schussler,, P. Macomber, and , M. Schiffman. 2003. Comparison between prototype hybrid capture 3 and hybrid capture 2 human papillomavirus DNA assays for detection of high-grade cervical intraepithelial neoplasia and cancer. J. Clin. Microbiol. 41: 4022– 4030.
12.
Chee, M.,, R. Yang,, E. Hubbell,, A. Berno,, X. C. Huang,, D. Stern,, J. Winkler,, D. J. Lockhart,, M. S. Morris, and , S. P. Fodor. 1996. Assessing genetic information with high-density DNA arrays. Science 274: 610– 614.
13.
Cheung, V. G.,, M. Morley,, F. Aguilar,, A. Massimi,, R. Kucherlapati, and , G. Childs. 1999. Making and reading microarrays. Nat. Genet. 21(1 Suppl.): 15– 19.
14.
Chevrier, D.,, S. R. Rasmussen, and , J.-L. Guesdon. 1993. PCR product by quantification by nonradioactive hybridization procedures using an oligonucleotide covalently bound to microwells. Mol. Cell. Probes 7: 187– 197.
15.
Chou, W.-H.,, F.-X. Yan,, D. K. Robbins-Weilert,, T. B. Ryder,, W. W. Liu,, C. Perbost,, M. Fairchild,, J. De Leon,, W. H. Koch, and , P. J. Wedlund. 2003. Comparison of two CYP2D6 genotyping methods and assessment of genotype-phenotype relationships. Clin. Chem. 49: 542– 551.
16.
Collins, M. L.,, B. Irvine,, D. Tyner,, E. Fine,, C. Zayati,, C. Chang,, T. Horn,, D. Anle,, J. Detmer,, L. Shen,, J. Kolberg,, S. Bushnell,, M. S. Urdea, and , D. D Ho. 1997. A branched DNA signal amplification assay for quantitation of nucleic acid targets below 100 molecules/ml. Nucleic Acids Res. 25: 2979– 2984.
17.
Compton, J. 1990. Nucleic acid sequence-based amplification. Nature (London) 350: 91– 92.
18.
Dicker, L. W.,, D. J. Mosure,, W. C. Levine,, C. M. Black, and , S. M. Berman. 2000. Impact of switching laboratory tests on reported trends in Chlamydia trachomatis infections. Am. J. Epidemiol. 151: 430– 435.
19.
Didenki, V. V. 2001. DNA probes using fluorescence resonance energy transfer (FRET): designs and applications. BioTechniques 31: 1106– 1121.
20.
Dovichi, N. J. 1997. DNA sequencing by capillary electrophoresis. Electrophoresis 18: 2393– 2399.
21.
Duggan, D. J.,, M. Bittner,, Y. Chen,, P. Meltzer, and , J. M. Trent. 1999. Expression profiling using cDNA microarrays. Nat. Genet. 21(1 Suppl.): 10– 14.
22.
Dyson, N. J. 1991. Immobilization of nucleic acids and hybridization analysis, p. 111– 156. In T. A. Brown (ed.), Essential Molecular Biology: a Practical Approach, vol. 2. IRL Press, Oxford, United Kingdom.
23.
Eisenstein, B. I. 1990. The polymerase chain reaction: a new method of using molecular genetics for medical diagnosis. N. Engl. J. Med. 322: 178– 183.
24.
Fahrlander, P. D., and , A. Klausner. 1988. Amplifying DNA probe signals: a “Christmas Tree” approach. Bio/ Technology 6: 1165– 1168.
25.
Fairfax, M. R. 1996. Evaluation of the Gen-Probe amplified Mycobacterium tuberculosis direct test. Am. J. Clin. Pathol. 106: 594– 599.
26.
Farthing, A.,, P. Masterson,, W. P. Mason, and , K. H. Vousden. 1994. Human papillomavirus detection by hybrid capture and its possible clinical use. J. Clin. Pathol. 47: 649– 652.
27.
Gelmini, S.,, C. Orlando,, R. Sestini,, G. Vona,, P. Pinzani,, L. Ruocco, and , M. Pazzagli. 1997. Quantitative polymerase chain reaction homogenous assay based on the use of fluorogenic probes for the measurement of c-erbB-2 oncogene amplification. Clin. Chem. 43: 752– 758.
28.
Griffin, H. G., and , A. M. Griffin. 1993. DNA sequencing. Recent innovations and future trends. Appl. Biochem. Biotechnol. 38: 147– 159.
29.
Guatelli, J. C.,, K. M. Whiffield,, D. Y. Kwoh,, K. J. Barringer,, D. D. Richman, and , T. R. Gingeras. 1990. Isothermal, in vitro amplification of nucleic acids by multienzyme reaction modeled after retroviral replication. Proc. Natl. Acad. Sci. USA 87: 1874– 1878.
30.
Hacia, J. G.,, L. C. Brody,, M. S. Chee,, S. P. Fodor, and , F. S. Collins. 1996. Detection of heterozygous mutations in BRCA1 using high density oligonucleotide arrays and twocolour fluorescence analysis. Nat. Genet. 14: 441– 447.
31.
Hankin, R. C. 1992. In situ hybridization: principles and applications. Lab. Med. 23: 764– 770.
32.
Haydock, P. V., and , S. A. Kochik. 1993. 3SR detection of Chlamydia trachomatis, p. 242– 246. In D. H. Persing,, T. F. Smith,, F. C. Tenover, and , T. J. White (ed.), Diagnostic Molecular Microbiology: Principles and Applications. American Society for Microbiology, Washington, D.C.
33.
Heid, C. A.,, J. Stevens,, K. J. Livak, and , P. M. Williams. 1996. Real time quantitative PCR. Genome Res. 6: 986– 994.
34.
Holland, P. M.,, R. D. Abramson,, R. Watson, and , D. H. Gelfand. 1991. Detection of specific polymerase chain reaction product by utilizing the 5′ to 3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA 88: 7276– 7280.
35.
Hubbard, R. A. 2003. Human papillomavirus testing methods. Arch. Pathol. Lab. Med. 127: 940– 945.
36.
Hughes, T. R.,, M. Mao,, A. R. Jones,, J. Burchard,, M. J. Marton,, K. W. Shannon,, S. M. Lefkowitz,, M. Ziman,, J. M. Schelter,, M. R. Meyer,, S. Kobayashi,, C. Davis,, H. Dai,, Y. D. He,, S. B. Stephaniants,, G. Cavet,, W. L. Walker,, A. West,, E. Coffey,, D. D. Shoemaker,, R. Stoughton,, A. P. Blanchard,, S. H. Friend, and , P. S. Linsley. 2001. Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer. Nat. Biotechnol. 19: 342– 347.
37.
Hunsaker, W R.,, H. Badri,, M. Lombardo, and , M. L. Collins. 1989. Nucleic acid hybridization assays employing dA-tailed capture probes. II. Advanced multiple capture methods. Anal. Biochem. 181: 360– 370.
38.
Iftner, R., and , L. L. Villa. 2003. Human papillomavirus technologies. J. Natl. Cancer Inst. Monogr. 31: 80– 88.
39.
Jonas, V.,, M. J. Alden,, J. I. Curry,, K. Kamisango,, C. A. Knott,, R. Lankford,, J. M. Wolfe, and , D. F. Moore. 1993. Detection and identification of Mycobacterium tuberculosis directly from sputum sediments by amplification of rRNA. J. Clin. Microbiol. 31: 2410– 2416.
40.
Kant, J. A. 1995. Direct DNA sequencing in the clinical laboratory. Clin. Chem. 41: 1407– 1409.
41.
Keiichi, K.,, C. Kamogawa,, M. Sumi,, S. Goto,, A. Hirao,, F. Gonzales,, K. Yasuda, and , S. Iino. 1999. Quantitative detection of hepatitis B virus by transcription-mediated amplification and hybridization protection assay. J. Clin. Microbiol. 37: 310– 314.
42.
Khan, J.,, M. L. Bittner,, Y. Chen,, P. S. Meltzer, and , J. M. Trent. 1999. DNA microarray technology: the anticipated impact on the study of human disease. Biochim. Biophys. Acta 1423: M17– M28.
43.
Kheterpal, I., and , R. A. Mathies. 1999. Capillary array electrophoresis DNA sequencing. Anal. Chem. 71: 31.– 37A.
44.
Konnick, E. Q.,, M. Erali,, E. R. Ashwood, and , D. R. Hillyard. 2005. Evaluation of the COBAS Amplicor HBV Monitor assay and comparison with the Ultrasensitive HBV Hybrid Capture 2 assay for quantitation of hepatitis B virus DNA. J. Clin. Microbiol. 43: 596– 603.
45.
Kwiatkowski, R. W.,, V. Lyamichev,, M. de Arruda, and , B. Neri. 1999. Clinical, genetic, and pharmacogenetic applications of the Invader assay. Mol. Diagn. 4: 353– 364.
46.
Kwoh, D. Y.,, G. R. Davis,, K. M. Whitfield,, H. L. Chappelle,, L. J. DiMichele, and , T. R. Gingeras. 1989. Transcription-based amplification system and detection of amplified human immunodeficiency virus type 1 with a bead-based sandwich hybridization format. Proc. Natl. Acad. Sci. USA 86: 1173– 1177.
47.
Kwok, S. 1990. Procedures to minimize PCR-product carryover, p. 142– 145. In M. A. Innis,, D. H. Gelfand,, J. J. Sninsky, and , T J. White (ed.), PCR Protocols: a Guide to Methods and Applications. Academic Press, Inc., San Diego, Calif.
48.
La Rocco, M. T.,, A. Wanger,, H. Ocera, and , E. Macias. 1994. Evaluation of a commercial rRNA amplification assay for direct detection of Mycobacterium tuberculosis in processed sputum. Eur. J. Clin. Microbiol. Dis. 13: 726– 731.
49.
Ledford, M.,, K. D. Friedman,, M. J. Hessner,, C. Moehlenkamp,, T. M. Williams, and , R. S. Larson. 2000. A multi-site study for detection of the factor V (Leiden) mutation from genomic DNA using a homogeneous invader microplate fluorescence resonance energy transfer (FRET) assay. J. Mol. Diagn. 2: 97– 104.
50.
Lee, L. G.,, C. R. Connel, and , W. Bloch. 1993. Allelic discrimination by nick translation PCR with fluorogenic probes. Nucleic Acids Res. 21: 3761– 3766.
51.
Levi, K., and , K. J. Towner. 2003. Detection of methicillinresistant Staphylococcus aureus (MRSA) in blood with the EVIGENE MRSA detection kit. J. Clin. Microbiol. 41: 3890– 3892.
52.
Lipshutz, R. J.,, S. P. Fodor,, T. R. Gingeras, and , D. J. Lockhart. 1999. High density synthetic oligonucleotide arrays. Nat. Genet. 21(1 Suppl.): 20– 24.
53.
Lipshutz, R. J.,, D. Morris,, M. Chee,, E. Hubbel,, M. J. Kozal,, N. Shah,, N. Shen,, R. Yang, and , S. P. Fodor. 1995. Using oligonucleotide probe arrays to access genetic diversity. BioTechniques 19: 442– 447.
54.
Little, M. C.,, J. Andrews,, R. Moore,, S. Bustos,, L. Jones,, C. Embres,, G. Durmowicz,, J. Harris,, D. Berger,, K. Yanson,, C. Rostkowski,, D. Yursis,, J. Price,, T. Fort,, A. Walters,, M. Collis,, O. Llorin,, J. Wood,, F. Failing,, C. O’Keefe,, B. Scrivens,, B. Pope,, T. Hansen,, K. Marino,, K. Williams, and , M. Boenisch. 1999. Strand displacement amplification and homogenous real-time detection incorporated in a second-generation DNA probe system, BDProbe TecET. Clin. Chem. 45: 777– 784.
55.
Livak, K.,, S. J. A. Flood,, J. Marmaro,, W. Giusti, and , K. Deetz. 1995. Oligonucleotides with fluorescent dyes at opposite ends provide a quencher probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Applic. 4: 357– 362.
56.
Loeffelholz, M. J.,, C. A. Lewinski,, S. R. Silver,, A. P. Purohit,, S. A. Herman,, D. A. Buonagurio, and , E. A. Dragon. 1992. Detection of Chlamydia trachomatis in endocervical specimens by polymerase chain reaction. J. Clin. Microbiol. 30: 2847– 2851.
57.
Marras, S. A. E.,, F. R. Kramer, and , S. Tyagi. 2002. Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. Nucleic Acids Res. 30: e122.
58.
Matsuzaki, H.,, S. Dong,, H. Loi,, X. Di,, G. Liu,, E. Hubbell,, J. Law,, T. Berntsen,, M. Chadha,, H. Hui,, G. Yang,, G. C. Kennedy,, T. A. Webster,, S. Cawley,, P. S. Walsh,, K. W. Jones,, S. P. Fodor, and , R. Mei. 2004. Genotyping over 100,000 SNPs on a pair of oligonucleotide arrays. Nat. Methods 1: 109– 111.
59.
Matthews, J. A., and , J. Kricka. 1988. Analytical strategies for the use of DNA probes. Anal. Biochem. 169: 1– 25.
60.
Mayer, S. P.,, J. Giamelli,, C. Sandoval,, A. S. Roach,, M. Fevzi Ozkaynak,, O. Tugal,, G. Rovera, and , S. Jayabose. 1999. Quantitation of leukemia clone-specific antigen gene rearrangements by a single-step PCR and fluorescence-based detection method. Leukemia 13: 1843– 1852.
61.
McCreedy, B. J., and , T. H. Callaway. 1993. Laboratory design and work flow, p. 149– 159. In D. H. Persing,, T. F. Smith,, F. C. Tenover, and , T J. White (ed.), Diagnostic Molecular Microbiology: Principles and Applications. American Society for Microbiology, Washington, D.C.
62.
Messing, J. 1993. M13 cloning vehicles: their contribution to DNA sequencing, p. 9– 22. In H. G. Griffin and , A. M. Griffin (ed.), DNA Sequencing Protocols. Humana, Totowa, N.J.
63.
Mullikin, J. C, and , A. A. McMurray. 1999. Sequencing the genome, fast. Science 283: 1867– 1868.
64.
Mullis, K.,, F. Faloona,, S. Scharf,, R. Saiki,, G. Horn, and , H. Erlich. 1986. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symp. Quant. Biol. 51: 263– 273.
65.
Mullis, K. B. 1990. The unusual origin of the polymerase chain reaction. Sci. Am. 262: 56– 65.
66.
Mullis, K. B., and , E A. Faloona. 1987. Specific synthesis of DNA in vitro via a polymerase-catalyzed reaction. Methods Enzymol. 155: 335– 350.
67.
Nelson, N. C., and , D. L. Kacian. 1990. Chemiluminescent DNA probes: a comparison of the acridinium ester and dioxetane detection systems and their use in clinical diagnostic assays. Clin. Chim. Acta 194: 73– 90.
68.
Nuwaysir, E. F.,, W Huang,, T. J. Albert,, J. Singh,, K. Nuwaysir,, A. Pitas,, R. Richmond,, T. Gorski,, J. P. Berg,, J. Ballin,, M. McCormick,, J. Norton,, T. Pollock,, T. Sumwalt,, L. Butcher,, D. Porter,, M. Molla,, C. Hall,, F. Blattner,, M. R. Sussman,, R. L. Wallace,, F. Cerrina, and , R. D. Green. 2002. Gene expression analysis using oligonucleotide arrays produced by maskless photolithography. Genome Res. 12: 1749– 1755.
69.
Orlando, C,, P. Pinzani, and , M. Pazzagli. 1998. Developments in quantitative PCR. Clin. Chem. Lab. Med. 36: 255– 269.
70.
Palva, A., and , M. Ranki. 1985. Microbial diagnosis by nucleic acid sandwich hybridization. Clin. Lab. Med. 5: 475– 490.
71.
Pease, A. C.,, D. Solas,, E. J. Sullivan,, M. T. Cronin,, C. P. Holmes, and , S. P. Fodor. 1994. Light-generated oligonucleotide arrays for rapid DNA sequence analysis. Proc. Natl. Acad. Sci. USA 91: 5022– 5026.
72.
Persing, D. H. 1991. Polymerase chain reaction: trenches to benches. J. Clin. Microbiol. 29: 1281– 1285.
73.
Persing, D. H. 1993. Diagnostic molecular microbiology: current challenges and future direction. Diagn. Microbiol. Infect. Dis. 16: 159– 163.
74.
Persing, D. H. 1993. In vitro nucleic acid amplification techniques, p. 51– 87. In D. H. Persing,, T. F. Smith,, F. C. Tenover, and , T. J. White (ed.), Diagnostic Molecular Microbiology: Principles and Applications. American Society for Microbiology, Washington, D.C.
75.
Persing, D. H., and , G. D. Cimino. 1993. Amplification product inactivation methods, p. 105– 121. In D. H. Persing,, T. F. Smith,, F. C. Tenover, and , T. J. White (ed.), Diagnostic Molecular Microbiology: Principles and Applications. American Society for Microbiology, Washington, D.C.
76.
Phear, G., and , J. Harwood. 1994. Direct sequencing of PCR products. Methods Mol. Biol. 31: 247– 256.
77.
Podzorski, R. P. 2004. Gel electrophoresis, Southern hybridization, and restriction fragment length polymorphism analysis, p. 273– 280. In D. H. Persing,, F. C. Tenover,, J. Versalovic,, Y.-W. Tang,, E. R. Unger,, D. A. Relman, and , T. J. White (ed.), Molecular Microbiology: Diagnostic Principles and Practice. American Society for Microbiology, Washington, D.C.
78.
Podzorski, R. P., and , D. H. Persing. 1993. PCR: the next decade. Clin. Microbiol. Newsl. 15: 137– 143.
79.
Podzorski, R. P., and , D. H. Persing. 1995. Molecular detection and identification of microorganisms, p. 130– 157. In P. R. Murray,, E. J. Baron,, M. A. Pfaller,, F. C. Tenover, and , R. H. Yolken (ed.), Manual of Clinical Microbiology, 6th ed. American Society for Microbiology, Washington, D.C.
80.
Podzorski, R. P., and , D. H. Persing. 1995. Molecular methods for the detection and identification of viral pathogens. J. Histotechnol. 18: 225– 232.
81.
Pollard-Knight, D.,, C. A. Read,, M. J. Downes,, L. A. Howard,, M. R. Leadbetter,, S. A. Pheby,, E. McNaughton,, A. Syms, and , M. A. W. Brady. 1990. Nonradioactive nucleic acid detection by enhanced chemiluminescence using probes directly labeled with horseradish peroxidase. Anal. Biochem. 185: 84– 89.
82.
Poulsen, A. B.,, R. Skov, and , L. V. Pallesen. 2003. Detection of methicillin resistance in coagulase-negative staphylococci and in staphylococci directly from simulated blood cultures using the EVIGENE MRSA detection kit. J. Antimicrob. Chemother. 51: 419– 421.
83.
Quesda, M. A. 1997. Replacement polymers in DNA sequencing by capillary electrophoresis. Curr. Opin. Biotechnol. 8: 82– 93.
84.
Raja, S.,, J. Ching,, L. Xi,, S. J. Hughes,, R. Chang,, W. Wong,, W. McMillian,, W. E. Gooding,, K. S. McCarty, Jr.,, M. Chestney,, J. D. Luketich, and , T. E. Godfrey. 2005. Technology for automated, rapid, and quantitative PCR or reverse transcription-PCR clinical testing. Clin. Chem. 51: 882– 890.
85.
Ranki, M.,, A. Palva,, M. Virtanen,, M. Laaksonen, and , H. Soderlund. 1983. Sandwich hybridization as a convenient method for detection of nucleic acids in crude samples. Gene 21: 77– 85.
86.
Rao, V. B. 1994. Direct sequencing of polymerase chain reaction-amplified DNA. Anal. Biochem. 216: 1– 14.
87.
Rapley, R. (ed.). 1996. PCR Sequencing Protocols. Humana, Totowa, N.J.
88.
Reddy, K. J., and , M. Gilman. 1993. Preparation of bacterial RNA, p. 4.4.1– 4.4.7. In F. M. Ausubel,, R. Brent,, R. E. Kingston,, D. D. Moore,, J. G. Seidman, and , K. Struhl (ed.), Current Protocols in Molecular Biology, vol. 1. John Wiley & Sons, New York, N.Y.
89.
Romano, J. W.,, B. van Gemen, and , T. Kievits. 1996. A novel, isothermal detection technology for qualitative and quantitative HIV-1 measurements. Clin. Lab. Med. 16: 89– 103.
90.
Ross, J. S. 1999. The impact of molecular diagnostic tests on patient outcomes. Clin. Lab. Med. 19: 815– 831.
91.
Ryan, D.,, B. Nuccie, and , D. Arvan. 1999. Non-PCR-dependent detection of the factor V Leiden mutation from genomic DNA using a homogenous invader microtiter plate assay. Mol. Diagn. 4: 135– 144.
92.
Ryncarz, A. J.,, J. Goddard,, A. Wald,, M. L. Huang,, B. Roizman, and , L. Corey. 1999. Development of a high-throughput quantitative assay for detecting herpes simplex virus DNA in clinical samples. J. Clin. Microbiol. 37: 1941– 1947.
93.
Saiki, R. K. 1989. The design and optimization of the PCR, p. 7– 22. In H. A. Erlich (ed.), PCR Technology: Principles and Applications for DNA Amplification. Stockton Press, New York, N.Y.
94.
Saiki, R. K.,, P. S. Walsh,, C. H. Levenson, and , H. A. Erlich. 1989. Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc. Natl. Acad. Sci. USA 86: 6230– 6234.
95.
Sanchez-Pescador, R.,, M. S. Stempien, and , M. S. Urdea. 1988. Rapid chemiluminescent nucleic acid assays for detection of TEM-1 β-lactamase-mediated penicillin resistance in Neisseria gonorrhoeae and other bacteria. J. Clin. Microbiol. 26: 1934– 1938.
96.
Sanger, F.,, S. Nicklen, and , A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463– 5467.
97.
Schachter, J. 1997. DFA, EIA, PCR, LCR, and other technologies: what tests should be used for diagnosis of chlamydia infections. Immunol. Investig. 26: 157– 161.
98.
Sia, I. G.,, J. A. Wilson,, M. J. Espy,, C. V. Paya, and , T. F. Smith. 2000. Evaluation of the COBAS AMPLICOR CMV MONITOR test for detection of viral DNA in specimens taken from patients after liver transplantation. J. Clin. Microbiol. 38: 600– 606.
99.
Southern, E.,, K. Mir, and , M. Shchepinov. 1999. Molecular interactions on microarrays. Nat. Genet. 21(1 Suppl.): 5– 9.
100.
Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol.. 98: 503– 517.
101.
Strickler, J. D., and , C. D. Copenhaver. 1990. In situ hybridization in hematology. Am. J. Clin. Pathol. 93(suppl.): 544– 548.
102.
Sun, X. W.,, A. Ferenczy,, D. Johnson,, J. P. Koulos,, O. Lungu,, R. M. Richart, and , T. C. Wright, Jr. 1995. Evaluation of the hybrid capture human papillomavirus deoxyribonucleic acid detection test. Am. J. Obstet. Gynecol. 173: 1432– 1437.
103.
Szollosi, J.,, S. Damjanovich, and , L. Matyus. 1998. Application of fluorescence resonance energy transfer in the clinical laboratory: routine and research. Cytometry 15: 159– 179.
104.
Tenover, F. C, and , E. R. Unger. 1993. Nucleic acid probes for detection and identification of infectious agents, p. 3– 25. In D. H. Persing,, T. F. Smith,, F. C. Tenover, and , T. J. White (ed.), Diagnostic Molecular Microbiology: Principles and Applications. American Society for Microbiology, Washington, D.C.
105.
Thejls, H.,, J. Gnarpe,, H. Gnarpe,, P. G. Larsson,, J. J. Platz-Christensen,, L. Ostergaard, and , A. Victor. 1994. Expanded gold standard in the diagnosis of Chlamydia trachomatis in a low prevalence population: diagnostic efficacy of tissue culture, direct immunofluorescence, enzyme immunoassay, PCR and serology. Genitourin. Med. 70: 300– 303.
106.
Trojan, L.,, A. Schaff,, A. Steidler,, M. Haak,, G. Thalmann,, T. Knoll,, N. Gretz,, P. Alken, and , M. S. Michel. 2005. Identification of metastasis-associated genes in prostate cancer by genetic profiling of human prostate cancer cell lines. Anticancer Res. 25: 183– 191.
107.
Tyagi, S.,, D. P. Bratu, and , F. R. Kramer. 1998. Multicolor molecular beacons for allele discrimination. Nat. Biotechnol. 16: 49– 53.
108.
Tyagi, S., and , F. R. Kramer. 1996. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14: 303– 308.
109.
Urdea, M. S.,, T. Fultz,, T. J. Anderson,, M. Running,, J. A. Hamren,, S. Ahle, and , C. A. Chang. 1991. Branched amplification multimers for the sensitive, direct detection of human hepatitis viruses. Nucleic Acids Symp. Ser. 24: 197– 200.
110.
Vogelstein, B., and , K. W. Kinzler. 1999. Digital PCR. Proc. Natl. Acad. Sci. USA 96: 9236– 9241.
111.
Walker, G. T.,, M. L. Fraiser,, J. L. Schram,, M. C. Little,, J. G. Nadeau, and , D. P. Malinowski. 1992. Strand displacement amplification—an isothermal, in vitro DNA amplification technique. Nucleic Acids Res. 20: 1691– 1696.
112.
Walker, G. T.,, M. C. Little,, J. G. Nadeau, and , D. D. Shank. 1992. Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system. Proc. Natl. Acad. Sci. USA 89: 392– 396.
113.
Watson, A.,, A. Mazumder,, M. Stewart, and , S. Balasubramanian. 1998. Technology for microarray analysis of gene expression. Curr. Opin. Biotechnol. 9: 609– 614.
114.
Wetmur, J. G. 1991. DNA probes: applications of the principles of nucleic acid hybridization. Crit. Rev. Biochem. Mol. Biol. 26: 227– 259.
115.
White, T. J. 1993. Amplification product detection methods, p. 138– 148. In D. H. Persing,, T. F. Smith,, F. C. Tenover, and , T. J. White (ed.), Diagnostic Molecular Microbiology: Principles and Applications. American Society for Microbiology, Washington, D.C.
116.
White, T. J.,, R. Madej, and , D. H. Persing. 1992. The polymerase chain reaction for the diagnosis of infectious diseases. Adv. Clin. Chem. 29: 161– 196.
117.
Wiedbrauk, D. L. 1992. Molecular methods for virus detection. Lab. Med. 23: 737– 742.
118.
Wolcott, M. J. 1992. Advances in nucleic acid-based detection methods. Clin. Microbiol. Rev. 5: 370– 386.
Tables
Introduction to Molecular Methodology
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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
/content/10.1128/9781555815905.ch05.ch05tab01
TABLE 1
Comparison of basic features of five real-time PCR instruments
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