Wittwer,Carl T

Wittwer,Carl T

Specific Aims

“Mutation scanning” techniques attempt to detect the presence of any sequence alteration in a fragment of DNA. When used with diploid DNA, mutation scanning commonly screens for differences between the two copies. The DNA fragments are generated by PCR and analyzed for completely matched hybrids called homoduplexes, and mismatched hybrids called heteroduplexes. Heteroduplexes are usually separated from homoduplexes on a separation matrix. Conventional scanning techniques are not homogeneous and require a separation step. We have recently introduced a homogenous scanning system that requires no separation or reagent addition after PCR (1). A simple dye is added before amplification, and a high-resolution melting curve to detect heteroduplexes requires only 1-2 minutes. Our current product, HR-1, is a single sample instrument, and uses a special dye, LCGreen I, that is not matched to common channels in real-time instrumentation. Our objectives are to identify a scanning dye that is compatible with standard optics, demonstrate that homogeneous scanning can be performed in microtiter format, and establish the sensitivity of SNP heterozygote detection in our new, high-throughput system.

1. Identify a robust DNA scanning dye compatible with standard fluorescein optics. Most dsDNA dyes (ethidium bromide, SYBR Green I) either inhibit PCR or do not detect heteroduplexes. We have recently synthesized and commercialized a new scanning dye (LCGreen I), but it has a spectrum that is not matched to standard fluorescein optics. We will test other commercially available dyes for the right spectral properties and the ability to detect heteroduplexes. Alternatively, we will chemically modify LCGreen I so that its excitation and emission wavelengths are similar to fluorescein.

1. Demonstrate homogeneous, closed-tube mutation scanning by high-resolution melting analysis in 384-well format. Our currently available closed-tube scanning instrument (HR-1, Idaho Technology) analyzes only one sample at a time. Each sample is manually placed in a small diameter capillary and is surrounded on all sides by a heating element for homogeneous temperature control and high-resolution acquisition. Most scanning applications are high-throughput, suggesting obvious advantages of a 96 or 384-well microtiter format. The LightTyper is currently manufactured by Idaho Technology and is a 96 or 384-well fluorescent imager that acquires melting curves with fluorescein-labeled probes for genotyping. Current temperature control and melting curve acquisition are “low resolution” compared to the HR-1. We will improve the resolution of the LightTyper so that heteroduplexes can be detected. The new dye identified in the first specific aim will allow us to use one set of optics for both scanning and genotyping.

1. Establish the sensitivity of SNP heterozygote detection according to amplicon size and base mismatch. We will use plasmids of 40, 50, and 60% GC content where one position is engineered to be either A, C, G, or T in order to construct all possible heterozygotes and homozygotes. Different size amplicons and different positioning of the primers in relation to the mismatch position will be systematically studied.

Our milestone for Phase II feasibility is a sensitivity of >90% for SNP detection in PCR products up to 300 bp using fluorescein optics and 384-well format.

Our long-term objective is to further develop homogeneous nucleic acid techniques, integrating sample preparation, amplification and analysis into cost effective research and clinical solutions.

Significance

Most genetic diseases are complex. Many different sequence alterations in the same or different genes result in the same disease phenotype. The initial hope that most human diseases are caused by a limited number of sequence alterations has turned out not to be true. Many genes are often involved, and many different mutations within a gene may cause the same or similar disease patterns. The future of genetic testing will require highly parallel analysis of many coding regions within the same gene and in many different genes.

The Human Genome Project has succeeded in sequencing most regions of human DNA. Work to identify the genes and sequence alterations associated with disease continues at a rapid pace. Usually, linkage studies associate phenotype with genetic markers like simple sequence repeats (SSRs) or single nucleotide polymorphisms (SNPs) to identify candidate genes. Then, specific sequence alterations including SNPs, insertions, and deletions that cause missense, frameshift, or splicing mutations pinpoint the gene and the spectrum of responsible mutations.

However, even when the genetic details become known, it is difficult to use this knowledge in routine medical practice because the methods to analyze DNA are expensive and complex. Only when costs are significantly lowered and the methods dramatically simplified will DNA analysis be used in every day clinical practice for effective disease detection and better treatment.

Sequencing is the gold standard for identifying sequence variation. Why not just sequence everything? Sequencing can be commercially feasible for genetic analysis – Myriad Diagnostics routinely sequences BRCA1 and BRCA2 for clients worldwide. The only drawbacks are cost (approaching $3,000 per individual) and complexity. Standard sequencing requires the following steps, 1) amplification by PCR, 2) clean up of the PCR product, 3) addition of cycle sequencing reagents, 4) cycle sequencing for dideoxy termination, 5) clean up of the termination products, and 6) separation on a DNA sequencer. This complexity can be automated, as it has been at Myriad and in all large sequencing centers. However, 90-99% of sequences come back normal. A simple method to identify sequences as normal could eliminate most of the time, cost, and effort of sequencing. These methods of screening DNA for abnormalities are known as “scanning” methods. Once an abnormality is identified, then and only then need it be sequenced or otherwise genotyped. Mutation scanning is in contrast to “genotyping” that focuses on detecting specific sequence alterations.

Many scanning techniques have been developed. These include single strand conformational polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), conformation sensitive gel electrophoresis (CSGE), denaturing high-performance liquid chromatography (dHPLC), and temperature gradient capillary electrophoresis (TGCE). Most of these methods are based on detecting heteroduplexes produced from the amplification of heterozygous DNA, that is, one chromosome copy is normal and the other is altered. The detection sensitivity ranges from 50-100% and depends on the PCR product length and type of mismatch (2). All of these techniques require separation on a gel or matrix and expose PCR products to the environment. Analysis either dilutes or eliminates the PCR product and either require hours (SSCP, DGGE, CSGE, TGCE), or analyzes only one sample at a time (dHPLC).

Most human mutations are present in one copy and can be detected by scanning techniques. Conventional scanning methods do not detect homozygous changes (SSCP is an exception, but the sensitivity is low). This is a limitation, because important homozygous mutations do occur in humans (e.g., cystic fibrosis F508del) and single copy organisms (bacteria, viruses) only have one copy. Homozygous changes are best identified by genotyping methods or sequencing. Sequencing also identifies heterozygous DNA, although interpretation is not always easy, especially for insertions or deletions.

This proposal aims to adapt a new heteroduplex detection technique to a highly parallel format in order to make screening DNA simple and cost effective. The method is simple because only PCR reagents and a nucleic acid dye are required. No separation or purification steps are necessary. After PCR, the 96- or 384-well plate is moved from the thermal cycling instrument to a high-resolution melting instrument and a melting curve is obtained in less than five minutes. The method is “closed-tube” with no reagent additions or risk of PCR contamination. The analysis does not dilute or destroy the PCR product. It can be used for any purpose after analysis.

High-resolution melting is similar to high-definition TV or enhanced satellite imaging. The ability to collect high-density information allows image magnification to reveal greater detail. The “images” of DNA-melting take the form of fluorescence vs temperature plots, or “melting curves”. Interpretation of data is greatly aided by software algorithms. Although DNA melting analysis has been known for many years, the conventional technique measures absorbance, requires large amounts of purified DNA, and takes hours to complete. In contrast, fluorescent melting curve analysis requires only a few minutes and can be performed directly on the PCR product mixture.

In addition to identifying the presence of a heterozygous change somewhere in the PCR product, high-resolution melting can often identify the specific mutation. In Preliminary Results, we show that all four common beta-globin heterozygotes (AS, AC, AE, SC) are distinguishable by the shape of their melting curves (3). In such cases, scanning and genotyping can be combined into one simple melting analysis. Obviously, since the number of possible variants is large, some sequence variants will show melting curves that are difficult or impossible to distinguish. For example, 300 different single base heterozygotes are possible within a 100 base region, and this does not consider, multiple base changes, insertions, or deletions. However, when the spectrum of mutations is limited high-resolution melting analysis will often genotype as well as scan. Sequencing can always be performed for confirmation, if the time and expense are indicated.

Not only can high-resolution melting be used for heteroduplex scanning and genotyping, it can identify homozygous sequence alterations as well. This is also covered in Preliminary Results, where the 4 common beta-globin homozygotes (AA, SS, CC, and EE) are distinguished. Again, not all homozygous changes will be distinguishable, but many will. The sensitivity and specificity of scanning and genotyping with high-resolution melting analysis depends entirely on the resolution of the technique. High-resolution melting analysis is easiest with a single sample where a capillary can be completely surrounded by a heating element. This proposal suggests that similar results can be obtained in micro-titer format that will be good enough for most applications. In Phase I, we will show that a 384-platform is good enough for heteroduplex scanning with an SNP detection sensitivity of at least 90% in PCR products up to 300 bp. In Phase II, we will improve detection sensitivity for heterozygous and homozygous variants, scan two interesting clinical targets, develop multiplexing strategies, and launch a commercial instrument.

Although not a commercial goal, our methods and heteroduplex dyes may also be used on conventional real-time PCR instruments. However, the resolution of these instruments for melting curve analysis is poor and the detection sensitivity will be limited (3). We also have broad protection on the methods through pending patent applications.

High-resolution scanning/genotyping has many applications in both gene discovery research and genetic testing. Although not a focus of the current application, the discrimination power of high-resolution melting analysis can be used as a genetic marker for initial disease correlation to identify responsible genes. The primary advantage of such a marker is that it is easy to obtain by rapid, non-destructive analysis without probes or electrophoresis. For example, loss of heterozygosity (LOH) could be established by melting curve analysis. Furthermore, the heterozygosity of the PCR product can be increased at will by including more than one variable locus, for example, multiple SNPs. The genotype does not need to be known for association studies; segregation with phenotype is the only requirement. PCR products could be equally spaced over a chromosome at areas of heterozygosity. No probes, no electrophoresis.

Once a gene is identified as a likely candidate responsible for a phenotype, all exons and splice junctions are screened for sequence variants that may be disease-causing mutations. Again, scanning techniques (and high-resolution melting) can be very useful to rule out normal exons. When an exon is abnormal, it must be followed up with sequencing to identify the specific polymorphisms and mutations that are present. This establishes the frequency and identity of various mutations and polymorphisms.

When a disease is caused by only a few mutations, direct genotyping tests are reasonable. These vary from conventional restriction digestion of PCR products to homogeneous fluorescent methods. However in most diseases, many mutations occur and specific genotyping tests are not feasible because too many would be required. The options are sequencing or scanning. We believe high-resolution melting can eliminate 90-99% of sequencing requirements in the analysis of complex genetic disease. So, are you still going to sequence everything? Consider this. Your first step in sequencing is to amplify by PCR. Can you spare 5 min to move your amplified PCR plate onto a melting instrument to scan for sequence variants? After 5 min, you can take your plate back and continue your normal sequencing workflow, including amplicon cleanup, addition of cycle sequencing reagents, thermal cycling for cycle sequencing, cleanup of your extension products, separation on a sequencer, and analysis of the results. Might you consider not going to sequencing for the 90-99% of your PCR products that are normal or can be directly genotyped by melting?

HIgh-resolution melting can easily determine the identity of individuals at highly polymorphic loci. In Preliminary Studies, we show that melting analysis of HLA-A exon 2 segregates members of a family into histocompatible groups. This is a very rapid and cost effective way to establish HLA identity between related individuals prior to transplantation. Similar HLA matching of unrelated individuals would be more complex but may be feasible. Forensic identity typing is also possible.

Another application of high-resolution melting that deserves brief mention is haplotyping of multiple polymorphisms. Different haplotypes produce hetero- and homoduplexes with different stabilities. Hence, the cis or trans sequence relationship results in distinguishable melting curves (as long as the loci are in the same melting domain). Haplotyping is difficult by other methods and impossible by many (e.g. sequencing).

A summary of high-resolution melting analysis compared to other scanning techniques follows.

High-Resolution Melting / Other Scanning Techniques
Method / Homogeneous / Separation-based
Amplicon Exposure / Closed-tube / Open environment
Time to Result / 1-5 min / Hours
Disposition of Sample / Reusable / Diluted, discarded
Application / Scanning and Genotyping / Scanning
Variants Detected / Hetero- and Homozygotes / Heterozygotes

Preliminary Studies

Instrumentation. We have been working on PCR methods and instruments for homogeneous DNA analysis for over 10 years. In the early 1990s, we developed rapid-cycle PCR, amplifying genomic DNA in as little as 10 min (). In the mid 1990s, STTR funding allowed us to develop the LightCycler (), one of the leading real-time PCR instruments available today. To date, approximately 4,000 LightCyclers have been sold. The R.A.P.I.D. (Ruggedized Advanced Pathogen Identification Device) is a modified LightCycler developed by Idaho Technology for the US military in 1998. It is rugged (still works after a 1 meter drop onto concrete), lightweight and small enough to fit into a backpack. Its intended use is as a defense against biologic warfare. Approximately 300 have been sold to date. In 2003, the R.A.P.I.D. was chosen as the platform for the Joint Biological Agent Identification and Diagnostics System (JBAID) of the US Government. The principal investigator of this proposal is the primary inventor of the LightCycler and related technologies, including the use of SYBR Green I, hybridization probes, and melting analysis in real-time PCR.

The LightTyper is a 96- or 384-well low-resolution melting instrument developed for genotyping with fluorescently-labeled probes that is currently manufactured at Idaho Technology and sold by Roche. High-resolution is not necessary because probe melting temperature transitions are broad and different genotypes are separated by several degrees C. The LightTyper competes against many other high-throughput SNP typing systems. Characteristics of the LightTyper will be detailed in the Experimental Plan. We will convert the LightTyper into a high-resolution instrument (codenamed the “LightScanner”) in order to achieve the specific aims of this grant.

The HR-1 is a high-resolution melting instrument that analyzes one sample at a time, launched commercially by Idaho Technology in the fall of 2003. The HR-1 was developed as a gold standard to see what might be possible with high-resolution melting. The sample geometry is ideal for temperature homogeneity because the sample capillary is completely surrounded by a heating element. Analysis is fast (1-2 min) with a throughput of 45 samples per hour and the price is right (<$10K), but the manual handling of fragile capillaries lessens its commercial appeal. The resolution obtainable on the HR-1 is vastly superior to the LightCycler and all other real-time PCR instruments. Further details on our instruments can be found on the Idaho Technology ( and Roche ( websites.

Probe melting analysis. Genotyping on the LightCycler or LightTyper monitors the melting of fluorescent probes. Thermal melting of DNA is a simple and elegant way to genotype and can be thought of as a “dynamic dot-blot”. Two strands of DNA fall apart or “melt” as the sample is gradually heated. The melting of hybridization probes was first observed on fluorescence vs. temperature plots acquired continuously during PCR (Fig. 1). In the annealing/extension phase, the probes hybridize to single stranded product and the fluorescence increases. When heating toward denaturation, the probes dissociate and the fluorescence returns to baseline levels. Exactly how they melt depends on the probe stability, and this depends on the genotype. Genotypes that differ in only a single base, can easily be discriminated, first demonstrated in 1997 for factor V Leiden (). The standard LightCycler scheme for genotyping uses 2 adjacent hybridization probes, one labeled 3' with fluorescein and the other labeled 5' with a longer wavelength dye (). In 2000, we modified this method to use only one single labeled probe (SimpleProbe), simplifying design considerations and cost. Recently, we developed a system that does not require any probes for genotyping. High-resolution melting of the entire amplicon after PCR allows genotyping without probes (,). Subtle differences in DNA sequence down to single base changes can be identified by high-resolution melting of the PCR product. The method can be applied to genotyping known mutations or to scanning for unknown sequence alterations within the PCR product. SNP genotyping (factor V Leiden) with adjacent hybridization probes (2 probes), SimpleProbes (1 probe) and high-resolution amplicon melting (no probes) is shown in Fig. 2.