October 15, 2005 (Vol. 25, No. 18)

Eliminating the Concern Over Running Out of DNA Samples Mid Experiment

Applications that need accurate replication often require large amounts of high-quality DNA for analysis and archiving. Areas where high fidelity, low mutation whole genome amplification (WGA) is necessary include molecular cloning, single cell analysis, and genome-wide analysis of single nucleotide polymorphisms (SNP).

“WGA takes one major concern out of the minds of genome-based drug discovery teams, i.e., the risk of running out of precious DNA samples in the midst of their study,” says Arumugham Ragunathan, Ph.D., sales development manager, Qiagen (www.qiagen.com). “Amplified DNA can be archived to revisit or share with other collaborators if and when necessary.”

Qiagen’s REPLI-g WGA is based on MDA (multiple displacement amplification). MDA is an isothermal (30oC), linear amplification using random hexamer primers and phi29 DNA polymerase.

Primers attach randomly to the template adding complementary base pairs to one strand, while displacing the other DNA strand. Primary products initiate secondary priming on the displaced DNA strand and continue displacing to create multiple branches. If the initial sample of genomic DNA is high quality, it can be amplified one million-fold while maintaining accurate loci and allele representation.

Qiagen’s phi29 DNA is a high processivity polymerase (processivity refers to the average number of bases the polymerase adds to a DNA strand being synthesized before the polymerase detaches from the template nucleic acid).

Low processivity polymerases synthesize fewer than 100 bases before they fall off the DNA template being replicated. As phi29 DNA polymerase moves along the DNA template, it remains attached displacing intervening primers causing multiple overlapping copies of the entire genome to be synthesized.

REPLI-g WGA can be performed directly on crude, lysed cells, whole blood, buccal swabs, and other biological samples; no amplified material has to be propagated in vivo. REPLI-g WGA also has low amplification bias at high amplification levels.

For example, an amplified nucleic acid greater than 10 kilobases generally has an error rate of about one in 106 to 107, compared with about three in 104 for PCR using TaqDNA polymerase and thermocycling, according to Dr. Ragunathan. PCR accumulates about one mutation per 900 bases after 20 cycles.

The phi29 DNA polymerase is “killed” at 65oC. The REPLI-g kit yields 45 micrograms (g) with a DNA concentration approximately 0.8 g per microliter (g/l). Further, the kit normalizes DNA across samples of different amounts: three samples, 1.0, 10, and 100 nanograms, respectively, all yield end products of 800 nanograms per milliliter (ng/ml).

Reforms Random Fragments

Rubicon Genomics’ (www.rubicongenomics. com) OmniPlex technology, GenomePlex, reformats random fragments of genomic DNA into plexisomes, which are defined size DNA fragments, structured to allow large regions of a genome to be amplified simultaneously from a single locus or multiple loci. These plexisomes, called OmniPlex libraries, can amplify the entire human genome in a single reaction and also use a high processive, high-fidelity DNA polymerase, according to the company.

Rubicon has extended Omni-Plex technology into two areas: whole methylome amplification (WMA), which amplifies only methylated DNA sequences, and whole transcriptome amplification (WTA), which amplifies total RNA.

“WGA, WMA, and WTA are related technologies for amplifying nanograms of starting genomic DNA or RNA by a thousand-fold in fewer than three hours,” notes John Langmore, Ph.D., vp of commercial development at Rubicon.

“WGA, WMA, and WTA produce amplified products that have been proven to perform as well or better than the template nucleic acids on most of the currently available genotyping, expression, and CGH platforms, including homogeneous assays such as TaqMan, and all the microarray and bead-based assays,” claims Dr. Langmore.

Rubicon WGA can correctly amplify limited samples with degraded DNA such as serum, plasma, CSF, buccal swabs, blood spots, urine and formalin-fixed biopsies, with no background, Dr. Langmore continues.

“Rubicon’s WGA, WMA, and WTA technologies can enable drug companies and institutions to discover novel drug targets from archived patient samples collected up to 50 years ago, as well as to evaluate effectiveness and toxicities for current drug development studies and clinical trials,” according to Dr. Langmore.

“Samples, typically collected before the genomics era, and used for purposes other than gene analysis and gene expression, are unique because they are linked with extensive data on disease or treatment outcomes, but their usefulness is limited because there is insufficient DNA or RNA, and the DNA and RNA are often highly degraded or contain PCR inhibitors.

“These limitations in sample quantity and quality can cause substantial frequencies of allele or locus drop-out as well as total sample failure,” Dr. Langmore asserts.

These technologies also allow amplification of DNA or RNA copy number and can dilute inhibitors in the sample if they are present.

“Genotyping, resequencing, methylation analysis, and abundance analysis of amplified materials are used to identify gene sequence, promoter methylation, and expression patterns associated with disease, which can lead to discovering new drug targets and predicting treatment outcomes,” adds Dr. Langmore.

Number of Challenges

Challenges include making WGA methods and tools higher throughput. Speed and simplicity are central issues in many applications, particularly diagnostics and high throughput screening.

Although Rubicon’s WGA, WMA, and WTA can all be completed in two to three hours using common general-purpose instruments, the company is reducing the time, volume, and steps to obtain products for microfluidic and point-of-care instruments.

Another challenge, according to Dr. Langmore, is to adapt the technologies to get more genetic, epigenetic, or expression information from individual cells.

“Although papers have been written showing the advantages of OmniPlex WGA for comparative genetic hybridization from individual sorted chromosomes and cells, considerable development work will be necessary to enable single cell pre-implantation or fetal diagnostics,” he continues.

The next steps for Rubicon’s technology include modifying the WGA process to detect gene methylation, for diagnosing cancer and other diseases from serum, urine, and other fluids, and for discovering drug targets and disease markers.

DNA methylation plays an important role in controlling gene expression and chromosome structure in mammalian cells. Genomic hypomethylation is associated with genomic disruption and chromosomal instability, whereas regions of specific DNA hypermethylation may be associated with inactivation of common tumor suppressor genes.

Cost and Time

“MethylPlex WMA, Rubicon’s whole methylome amplification process, has cost and time advantages over other methylation assays, which require bisulfite degradation of DNA, are not reproducible, and are difficult to automate,” says Dr. Langmore.

For automation, WGA, WTA, and WMA protocols are streamlined to produce amplified nucleic acids in two to three hours. Other improvements seen regarding WGA, WTA, and WMA are protocols to allow library amplification isothermally rather than by thermal cycling.

“WGA and WTA will find new applications in biomedical research and diagnostics as a combined process of total nucleic acid amplification,” according to Dr. Langmore. “This is being practiced in at least one diagnostics laboratory, which is simultaneously amplifying DNA and RNA for microarray detection and characterization of DNA and RNA viruses using a single microarray.”

Rubicon has also tested simultaneous amplification of DNA, methylated DNA, and RNA for analyses of all three types of genetic information from the same small patient sample. Third parties are testing WGA, WMA, and WTA on point-of-care diagnostic devices, which could produce patient test results in an hour or under.

Research Services

Asterand (www.asterand.com) is a research service company helping customers perform research with human tissue and associated clinical data, provided by the company’s international network of collaborators. In building these collaborations, Asterand, which merged with Pharmagene (www.pharmagene.com) last month as a result of a reverse takeover, has identified populations with high incidences of certain diseases, such as type 2 diabetes, which appears to have a strong genetic line. The company collects DNA samples from affected families in this population for diabetes researchers.

“While it is easy to collect blood from affected individuals, who have blood drawn regularly in the clinic to measure glucose levels,” explains James Eliason, Ph.D., CSO at Asterand, “healthy family members are not always willing to come to the clinic to give blood samples.”

In the latter case, Asterand collects buccal cells using a mouthwash kit. Since cell numbers in mouthwash are much lower compared with a standard blood draw, WGA is used to ensure sufficient DNA for research.

“WGA allows much more genetic research in instances where access to a large number of cells is limited, and in cases where individuals have a high risk for a disease such as cancer but are currently healthy,” says Dr. Eliason. “WGA has advantages compared with immortalizing lymphocytes with Epstein-Barr virus, which is time consuming and has a variable success rate.”

WGA is also useful with laser capture microdissected tissue. Asterand is working with fixed tissues like those used for standard diagnoses in hospitals, which are kept in hospital archives.

These older samples can be useful for cancer research where patient outcome results are needed. Also, there is a tendency for cancer patients to be diagnosed based upon needle biopsies, where only a small sample is taken. Patients are then given neoadjuvant therapy before surgery, so only a portion of that small biopsy sample may be available for research.

“To get faithful representation of the whole genome, one still needs on the order of thousands of cells, minimally. It will require more work to push that limit lower for routine analyses,” according to Dr. Eliason.

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