The death of PCR for sequencing amplification?
PCR (polymerase chain reaction) amplification for sequencing
applications and dramatically increase the efficiency of sample
preparation.
The methods pave the way for cost-effective sequencing of individual human genomes enabling large numbers of genomic to regions to be extracted from a sample before sequencing enabling researchers to sequence only the genomic regions of interest. The new techniques use microarray gene chips to capture and enrich specific genomic sequences for direct resequencing, enabling researchers to discover mutations in specific genes more efficiently than ever before. The first method, described in two early access articles in the journal Nature Methods, uses a 'microarray-based genomic selection' method that removes the need for whole-genome amplification using PCR techniques that can bias sequence results and lead to mutations being missed. "This new technology will replace polymerase chain reaction (PCR) for many purposes," said Dr Richard Gibbs, director of the Human Genome Sequencing Center (HGSC) at Baylor College of Medicine and senior author of one the reports entitled 'Direct selection of human genomic loci by microarray hybridization'. "If the aim is to sequence a whole genome for everybody, this is a huge step in that direction." The authors showed that the technique could be used to discover variation in the human BRCA1 that have been implicated in a number of hereditary cancers, such as breast, ovarian and prostate cancer. The researchers captured target sequences from Burkitt's Lymphoma cell line NA047671 DNA cell line using high-density oligonucleotide microarrays custom-made by Nimblegen. "You take the DNA and you hybridise it on the chip," said Dr George Weinstock, co-director of the HGSC and contributing author. "Then you wash away everything that doesn't stick. This can enrich the portion of the genome to be studied by factors of three hundred or more." After the non-hybridised materials were removed from the array, the captured sequences were sequenced using Roche / 454 Life Sciences' Genome Sequencer FLX. The researchers found that the data generated were highly specific and that the downstream DNA sequencing steps were 'consistently superior' to average performance using non-captured DNA sources. The authors attribute these improvements to the capture-enrichment process removing impurities that can cause problems during the first emulsion PCR step of the 454 sequencing process. The second report, entitled 'Microarray-based genomic selection for high-throughput resequencing' used the MGS technique to capture and resequence two X chromosome-linked genomic regions. The researchers showed that large human genomic regions on the order of hundreds of kilobases can be enriched and resequenced. They believe that when combined with next-generation sequencing technologies such as the 454 FLX and Illumina's Genome Analyser large-scale resequencing in single-investigator laboratories would be possible at levels comparable to conventional genome sequencing centres. Another approach to multiplex amplification is also described in Nature Methods in a paper entitled 'Multiple amplification of large sets of human exons' that uses programmable microarrays to release the oligonucleotides used to capture and amplify the DNA fragments. The programmable microarray synthesises a library of probes that consist of a universal 30 nucleotide motif flanked by 20 nucleotide 'targeting arm' segments that are designed to hybridise immediately up- and downstream of a specific genomic target. While the system worked with very high specificity, the capture and amplification of target sequences was not uniform. They postulate that one of the reasons for this could be the hybridisation of the genomic DNA is inefficient due to the low concentration of targeting oligos on the arrays. The researchers write that: "although this method has great potential, improving the uniformity with which individual targets are captured and amplified remains crucial."
