Advances in Nucleic Acid Detection and

Biochemical Society Focused Meeting held at Hinxton Hall, Cambridge, U.K., 28–29 October 2008. Organized and Edited by Simon Baker (Oxford Brookes, U.K.), Jeremy Gillespie (Thermo Fisher Scientific, U.K.), Simon Hughes (Oxford Gene Technology, U.K.), Ian Kavanagh (Thermo Fisher Scientific, U.K.) and Devin Leake (Thermo Fisher Scientific, U.S.A.).

Advances in Nucleic Acid Detection and Quantification Ian C. Kavanagh*1 and Simon C. Baker† *Thermo Fisher Scientific, ABgene House, Blenheim Road, Epsom KT19 9AP, U.K., and †School of Life Sciences, Oxford Brookes University, Gypsy Lane, Oxford OX3 0BP, U.K.

Abstract The last decade has seen many changes in molecular biology at the bench, as we have moved away from a primary goal of cataloguing genes and mRNA towards techniques that detect and quantify nucleic acid molecules even within single cells. With the invention of the polymerase chain reaction (PCR), a nucleic acid sequence could now be amplified to generate a large number of identical copies, and this launched a new era in genetic research. PCR has developed in parallel to fluorescent hybridization probing to provide low-, mediumand high-throughput detection methods. However, PCR and hybridization detection have significant drawbacks as long-term solutions for routine research and diagnostics assays. Therefore many novel methods are being developed independently, but as yet no one technique has emerged as a clear replacement for PCR, microarrays or even sequencing. In order to examine the technological horizon in this area, around 90 delegates assembled at Hinxton Hall, Cambridge, U.K. on 28 and 29 October 2008 for a Biochemical Society/Wellcome Trust Focused Meeting sponsored by Thermo Fisher Scientific and the British Library. The title of the meeting was ‘Advances in Nucleic Acid Detection and Quantification’, and the primary aim was to bring together scientists from different disciplines who nevertheless are trying to develop reliable methods for the quantification or detection of RNA and DNA molecules. This meant that physical and organic chemists, microbial ecologists and clinicians appeared alongside molecular biologists. An introductory session on general nucleic acid detection technologies was initiated with a fascinating insight into single-molecule, singlecell hybridization from Professor Sir Edwin Southern. This served as an ideal base for sessions on single-cell molecular biology and an examination of current applications of emerging technology. This issue of Biochemical Society Transactions contains some of the papers prepared by speakers at the meeting, and highlights not only how PCR and microarrays are already being replaced, but also which methods are likely to replace them.

Introduction The last decade has seen many changes in molecular biology at the bench. We have moved away from a primary goal of cataloguing genes and mRNA towards techniques that detect and quantify nucleic acid molecules even within single cells. Key words: DNA, label-free detection, PCR, Raman spectroscopy, RNA, single-cell analysis. Abbreviations used: COLD-PCR, co-amplification at lower denaturation temperature-PCR; ffDNA, free fetal DNA; HRM, high-resolution melting; MDA, multiple displacement amplification; PGD, pre-implantation genetic diagnosis; qPCR, quantitative PCR; RT, reverse transcription; WGA, whole genome amplification. 1 To whom correspondence should be addressed (email ian.kavanagh@thermofisher.com).

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Advances in Nucleic Acid Detection and Quantification


Quantification and detection of nucleic acid continues to evolve as new technologies become accessible to researchers from many disciplines. As we approach the 25th anniversary of the conception of the polymerase chain reaction (PCR), changes in both PCR-based and hybridization-based technologies can be anticipated. Although the development of precise techniques such as hybridization microarrays and quantitative (or real-time) PCR has enabled a new understanding of gene expression, the shortcomings of these established techniques are beginning to become apparent. Therefore it is prudent to look to new technologies which C The

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will combine or enhance the best attributes of existing protocols.

PCR in the detection and quantification of nucleic acids With the invention of PCR by Kary Mullis and co-workers [1], a nucleic acid sequence could now be amplified in a cyclic process to generate a large number of identical copies. The ability to readily analyse these products launched a new era in genetic research that has dramatically advanced the ability to detect and quantify nucleic acids. Indeed, Morling [2] explains how PCR-based methods have revolutionized the forensic genetics field for DNA analysis, so that the advances over the last 20 years now allow informative routine investigations, as well as more advanced special investigations in cases concerning crime, paternity, relationship and disaster victim identification. The functionality of PCR makes it possible to use DNA investigations extensively in forensic genetics because minute amounts of DNA from biological materials (100 pg of DNA or less) can be used in the analysis. This allows genetic typing or identification of SNPs (single-nucleotide polymorphisms) by amplifying stretches of DNA to detect VNTRs (variable numbers of tandem repeats) or STRs (short tandem repeats) [2]. Evolving from the standard PCR technique, qPCR (quantitative PCR) is a technique used to amplify and simultaneously quantify the target sequence of a DNA or RNA molecule [3,4]. qPCR has a wide range of applications in many biological disciplines, enabling detection and quantification of gene expression levels, DNA copy number, viral titres and transgene copy number, as well as allelic discrimination, verification of microarray data and siRNA (short interfering RNA) knockdown validation. The increase in the use of this method over the last 10 years has reached the point where qPCR instruments are now commonplace in biochemistry research laboratories, which has resulted in the emergence of many novel applications to capitalize on this. One such application is HRM (high-resolution melting), which detects DNA sequence variation by measuring the changes to the melting temperature of a double-stranded PCR amplicon [5,6]. Taylor [7] investigated the use of HRM as a technique for successful mutation scanning, in order to detect sequence variants without the need for prior knowledge of the identity or precise location of the variant. HRM was shown to have high sensitivity (low number of false negatives) and high specificity (low number of false positives), with performance as good as, or better than, other commonly used mutationscanning techniques. Among other benefits of HRM is the ability to perform all assays under identical thermal cycling conditions (for improved ease of use), in a closed system (to reduce amplicon contamination), with a shorter turnaround time and at lower cost per reaction than other techniques. Another technique developed recently uses a new form of PCR that preferentially enriches ‘minority alleles’ from mixtures of wild-type and mutation-containing sequences, C The

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termed COLD-PCR (co-amplification at lower denaturation temperature-PCR) [8]. Li and Makrigiorgos [9] described how replacing regular PCR with COLD-PCR yields products containing high percentages of variant alleles, thus permitting their detection using downstream detection techniques. Since PCR is the initial step for almost all genetic analysis, COLD-PCR could provide a general platform to improve the sensitivity of many of the DNA-variation-detection technologies, including Sanger sequencing, pyrosequencing, single-molecule sequencing, mutation scanning, mutation genotyping and methylation assays. Similarly, with advances in thermal cycler and real-time detection technology, the costs have now been driven down to levels where it is becoming feasible to use PCR or qPCR for the diagnosis of disease in the developing world. Huggett et al. [10] discussed, with specific reference to tuberculosis, the current status of nucleic acid amplification techniques in a developing world setting and examined the proposed benefits of improving disease diagnosis. This highlights that a quick, reliable diagnosis leading to prompt and appropriate treatment is possible, but even so, the assay development currently lags behind that for HIV and malaria.

Single-cell genetics It is well known that, within a population of genetically identical cells, there will be a significant variation in the expression of any given gene or genes [11]. Similarly, the very nature of sampling cells from a three-dimensional tissue can often lead to errors in measurements because of the averaging effect of including non-representative cells. It has therefore become clear that data obtained from a population of cells are not reflective of the individual, and so cells should be analysed as a single entity for accurate biochemical measurements. This represents a significant challenge to current detection technologies, because of the limited amount of nucleic acid that is present in a single cell. Lasken [12] discussed how WGA (whole genome amplification) methods from limiting specimens and environmental samples can generate the sufficient amounts of DNA that are frequently required for use in genomic analysis. Specifically, the author described how the MDA (multiple displacement amplification) reaction is increasingly the method of choice for many applications because of its extensive coverage of the genome, the generation of extremely long DNA products compared with other WGA methods, and the high DNA yields, even from exceedingly low amounts of starting material. Although WGA is well suited for use in downstream assays for qualitative detection, amplification bias is an important factor to consider where the goal is to precisely quantify the relative amounts of DNA present in a sample. Nevertheless, the MDA reaction has been demonstrated to have the lowest amplification bias when compared with many of the other WGA methods, as shown by Handyside et al. [13] who used WGA for single-cell analysis in PGD (pre-implantation genetic diagnosis) following assisted conception. PGD


typically relies on the genetic analysis of the first and second polar bodies and/or one or two single blastomeres biopsied from the fertilized zygote or cleavage-stage embryo respectively. With such limited genetic information available from a single cell, there is a high susceptibility to errors owing to unsuitable and inaccurate detection techniques and stringent precautions are essential throughout to avoid misdiagnoses. However, the use of isothermal WGA, unlike previous PCR-based methods, provided sufficient DNA for diagnosis of any known single-gene defect by standard methods under normal laboratory conditions [13]. A companion to PGD is non-invasive prenatal diagnosis, which is now also a clinical reality, following the detection of ffDNA (free fetal DNA) in the maternal circulation [14]. Maddocks et al. [15] discuss that apoptosis of the placenta (more precisely the syncytiotrophoblast cells of the chorionic villi) is the major source of ffDNA in the maternal plasma and how these minute amounts of ffDNA are being targeted for diagnostic assays to eliminate the need for invasive techniques such as amniocentesis. The group is part of the SAFE (Special Non-Invasive Advances in Fetal and Neonatal Evaluation) NoE (Network of Excellence), which was established to cover primary scientific research, ethics and socio-economic areas within the arena of fetal and neonatal wellbeing [15]. Whereas the majority of the SAFE project’s genetic analyses utilizes PCR-based techniques, a recent study has used a shotgun approach with next-generation sequencers to completely characterize sequences of ffDNA [16]. Although this study was very promising, it was only performed on a small number of samples, which is indicative of the high-cost and labour-intensive processes currently associated with these sequencers. However, with the rapid improvement in technology, the increase in throughput and decrease in time to achieve results could result in future routine application of these technologies in both research and diagnostic laboratories. Studying the transcriptome of a single cell or a very small biological sample usually requires RNA in larger quantities than is available, especially when using techniques such as microarray expression profiling. Consequently, amplification methods have also been developed for transcriptome RNA. However, these methods are still susceptible to bias and complex pre-amplification reactions may alter the relative abundance of specific transcripts and thus yield erroneous results in the subsequent analyses [17]. Bengtsson et al. [18] have recently reported a single-cell RT (reverse transcription)–qPCR protocol that requires no purification, but maintains mRNA integrity and demonstrates no inhibition of the RT and PCR steps. This study also investigated the nature of noise in RT–qPCR and concluded that variation for abundantly expressed genes is negligible, when compared with the normal biological variation occurring between cells, but measurements of rare transcripts are likely to be complicated by the limitations of PCR. Day [19] described a systems biology approach to single-cell biomarker quantification, using microfluidic PCR coupled with flow-assisted cell sorting, which was termed a μTAS (micro total analytical

system). The potential for analysing individual cells on a miniaturized scale suggest this approach could readily provide a basis for molecular biomarker quantification. An alternative approach to this is to tightly couple a probe hybridization event to the release of nucleic acid from a cell. This elegant combination of lysis and hybridization in situ, along with high-definition image processing, have allowed Southern and co-workers (http://www.ogt.co.uk/) to overcome some of the amplification-related problems of working with single cells. The method allows individual labelled molecules from single cells to be counted by fluorescence without further amplification. By arraying tens of thousands of individual cells, it will be possible to determine mRNA levels of up to 50 genes in a population.

Emerging technologies Fluorescent dyes have played an important part in the development of both solution-state DNA amplification methods (such as qPCR) and immobilized hybridization applications (microarrays). To date, the advantages in ease of use, multiplexing and low perceived toxicity of dyes have outweighed their disadvantages. However, it has long been recognized that bulky dyes attached to nucleotides or oligonucleotides have a steric effect during extension and hybridization with nucleic acids. As DNA-detection methods develop to allow the detection of single molecules, the influence of these bulky dye conjugates become progressively more significant. Ultimately, the goal for nucleic acid detection and quantification will be a label-free method, and several groups are working towards this goal. Up to now, label-free methods have focused on detection of hybrids such as those found in microarray analysis. A variety of spectroscopic techniques have been used to assay the formation of duplex nucleic acid, of which Raman spectroscopy shows considerable promise and has been used in the characterization of populations of DNA sequences [20]. Although discernible against a background of cellular constituents, the Raman signal specific for DNA is weak, as are spectra from most other compounds. To compensate for this, both gold and silver nanoparticles have been employed successfully to enhance the signal [21–24]. The use of gold or silver nanoparticles in conjunction with metal ions (e.g. iron or ruthenium) or metal–polymer mediators has also led to electrochemical detection of hybridization. A huge variety of arrangements of DNA-binding compounds and mediators have been explored, using detection by Raman spectroscopy, surface plasmon resonance or atomic force microscopy [25,26]. Miniaturized detectors suitable for ultra-microarrays are dominated by these physicochemical methods, while the more indirect detection afforded by biosensors does not seem to have the potential for widespread uptake. Liao et al. [27] have pioneered an electrochemical DNA-detection system mediated by horseradish peroxidase, but the ability of the newer physicochemical methods to detect the hybridization of single molecules without enzyme-mediated amplification looks set to dominate the research effort. C The


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Solution-state label-free detection of specific nucleic acid sequences has lagged behind the advances made in the conjugated systems exemplified by microarrays. However, work at Aston University [28,29] has led to the development of a fibre-optic-based system. A DNA probe is bound to a longperiod fibre-grating and hybridization in solution is detected by changes in the light path as the hybridization event occurs. If the system can be made sufficiently robust, it may open the way for new accurate point-of-care applications, which currently use inhibitor-susceptible enzyme-based dipsticks. In conclusion, we anticipate that the next 5–7 years will see further changes in the way nucleic acids are quantified and detected. The papers gathered in this issue give an indication of the breadth of effort and the multidisciplinary nature of current research. While complete consideration of their implications is outside the scope of this article, it is also likely that the division between the current roles played by qPCR, microarrays and sequencing will become increasingly indistinct.

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Received 9 January 2009 doi:10.1042/BST037e001