Mark G. Herrmann

Continuous Fluorescence Monitoring of Rapid Cycle DNA Amplification

Rapid cycle DNA amplification was continuously monitored by three different fluorescence techniques. Fluorescence was monitored by (i) the double-strand-specific dye SYBR Green I, (ii) a decrease i...

14 – Rapid thermal cycling and PCR kinetics

Publisher Summary DNA amplification by polymerase chain reaction (PCR) can be performed rapidly. Rapid temperature cycling for PCR, last reviewed in 1994, is defined as completing 30 cycles of amplification in <30 min. By using high surface area to volume sample containers and circulating air for heating and cooling, amplification in 15 min or less is readily achieved. This chapter discusses the use and advantages of rapid thermal cycling for PCR. One of the advantages of rapid thermal cycling for PCR is that it minimizes the amplification time and is thus useful when multiple sequential amplifications are required. Various instruments and protocols related to the process are also described along with several illustrative figures and tables. Other specialized instruments for rapidly changing temperature and for continuous monitoring of PCR by fluorescence are mentioned too. Moreover, the chapter also discusses the applications of rapid thermal cycling. PCR kinetics is outlined separately for clarity. The rates of the reactions that occur during PCR are dependent on temperature and can be individually measured. These include both productive reactions and nonproductive reactions.

Multiplex genotyping by melting analysis of loci-spanning probes: β-globin as an example

Multiplexing genotyping technologies usually require as many probes as genetic variants. Oligonucleotides that span multiple loci--loci spanning probes (LSProbes)--hybridize to two or more noncontiguous DNA sequences present in a template and can be used to analyze multiple variants simultaneously. The intervening template sequence, omitted in the LSProbe, creates a bulge-loop during binding. Melting temperatures of the probe, monitored by fluorescence reading are specific to the presence or absence of the mutations. We previously described LSProbes as a molecular haplotyping tool and apply here the principle to genotype simultaneously three mutations of the beta-globin gene responsible for the corresponding hemoglobinopathies. Analysis with both labeled and unlabeled LSProbes demonstrate that the four possible alleles studied (WT, HbS, HbC, and HbE) are identifiable by the specific melting temperatures of the LSProbes. This demonstrates that, in addition to their haplotyping capabilities, LSProbes are able to genotype in a single step, loci 58 nucleotides apart.

Instrument Comparison for Dna Genotyping by Amplicon Melting

DNA melting analysis for the identification of sequence is increasingly used in molecular diagnostics. Recent advances in DNA melting analysis, including high-resolution instrumentation and specialized fluorescent DNA-binding dyes, allow genotyping by whole amplicon melting without probes. With the popularity of melting analysis as a diagnostic tool, there is a need to characterize the ability of commercially available real-time PCR instruments to perform high-resolution amplicon melting analyses. Four real-time instruments varying in sample format, throughput, and heat transfer (Cepheids SmartCycler, Idaho Technologys HR-1, and Roches LightCycler 1.2 and LightCycler 2.0) were evaluated for their ability to differentiate homozygous genotypes at the human β-globin sickle cell locus. The melting transition was monitored by including the dye LCGreen Plus in the PCR, and the data were uniformly analyzed with custom in-house software. The wild-type and mutant homozygous genotypes differed by a theoretical Tm of 0.09°C and were best discriminated by the high-resolution HR-1 instrument. All instruments could identify a double single nucleotide polymorphism heterozygote by the heteroduplexes formed. However, signal-to-noise ratios varied from 260 to 3500, suggesting that melting instrument design (data acquisition, data density, thermal control) determines the accuracy of genotyping by amplicon melting. (JALA 2006;11:273-7)

Genotyping β-globin Mutations (Hb S, Hb C, Hb E) by Multiplexing Probe Color and Melting Temperature

Human β-globin has over 50 known mutations in exon 1 causing various hemoglobinopathies (1,2). The mutations include base pair substitutions, deletions and splicing defects. Hemoglobin S and C, (codon 6) and E (codon 26) are base pair substitutions that occur often enough to allow for routine screening (Hb S occurs at an allele frequency of 1:400 in African American’s)(3). High performance liquid chromatography (4) and isoelectric focusing (5) are routinely used for primary patient screening. However, phenotypic screening does not always necessarily coincide with the genotype, for example Hb S, Hb GNorfolk and HbFort-de-France all have the same isoelectrofocusing point (6). Genotyping can be done by allele specific amplification (7), DNA amplification followed by restriction digestion (8) or by fluorescently labeled allele specific primers and gel based detection with color photography (9). These methods require hours to days for completion. Recently, genotyping by determining the melting temperature (Tm) of hybridized fluorescently-labeled oligonucleotide probes was reported (10,11). We have extended the power of this technique by multiplexing different colored probes to simultaneously genotype multiple alleles (12). This procedure has been applied to homogenous genotyping of hemoglobin S, C, and E in a single tube by melting curve analysis by using different colored probes and Tm multiplexing.