Whole genome amplification: Difference between revisions
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In many fields of research, including [[preimplantation genetic diagnosis]], [[cancer research]] or [[forensic medicine]], the scarcity of genomic [[DNA]] can be a severely limiting factor on the type and quantity of genetic tests that can be performed on a sample. One approach designed to overcome this problem is '''whole genome amplification''' (WGA). The objective is to amplify a limited DNA sample in an aspecific way, in order to generate a new sample that is indistinguishable from the original but with a higher DNA concentration. The ideal WGA technique would amplify a sample up to a microgram level while respecting the original sequence representation. | In many fields of research, including [[preimplantation genetic diagnosis]], [[cancer research]] or [[forensic medicine]], the scarcity of genomic [[DNA]] can be a severely limiting factor on the type and quantity of genetic tests that can be performed on a sample. One approach designed to overcome this problem is '''whole genome amplification''' (WGA). The objective is to amplify a limited DNA sample in an aspecific way, in order to generate a new sample that is indistinguishable from the original but with a higher DNA concentration. The ideal WGA technique would amplify a sample up to a microgram level while respecting the original sequence representation. | ||
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Latest revision as of 17:29, 20 August 2012
In many fields of research, including preimplantation genetic diagnosis, cancer research or forensic medicine, the scarcity of genomic DNA can be a severely limiting factor on the type and quantity of genetic tests that can be performed on a sample. One approach designed to overcome this problem is whole genome amplification (WGA). The objective is to amplify a limited DNA sample in an aspecific way, in order to generate a new sample that is indistinguishable from the original but with a higher DNA concentration. The ideal WGA technique would amplify a sample up to a microgram level while respecting the original sequence representation.
PEP and DOP-PCR
The first WGA methods were described in 1992, and were based on the principles used in the Polymerase Chain Reaction (PCR) technique. Zhang and coworkers developed the primer extension PCR (PEP) and Telenius and collaborators designed the degenerated oligonucleotide primed PCR (DOP-PCR) (Telenius et al., 1992; Zhang et al., 1992).
- PEP involves a high number of PCR cycles; using Taq polymerase and 15 base random primers that anneal at a low stringency temperature. Although the PEP protocol has been improved in different ways (improved-PEP, I-PEP) (Dietmaier et al., 1999), it still results in incomplete genome coverage, failing to amplify certain sequences such as repeats, induces an amplification bias of the order of 103 to 106 (Xu et al., 1993; Sermon et al., 1996; Dean et al., 2002; Jiao et al., 2003) and has a limited efficiency on very small samples (such as single cells). Moreover, the use of Taq polymerase implies that the maximal product length is about 3 kb. Although PEP has been evaluated on single blastomeres and has even been applied during PGD for beta-thalassemia, it has been suggested to use PEP only in selected cases as the amplification conditions should be modified for each particular disorder (Xu et al., 1993; Sermon et al., 1996; Jiao et al., 2003).
- DOP-PCR is a well-established, widely accepted and technically straightforward method. It has been applied to single or small pools of cells and in PGD, as for instance for comparative genomic hybridization (Wells and Delhanty, 2000; Wells et al., 2002; Fragouli et al., 2006); recycling of fixed blastomeres (He et al., 1999) and loss of heterozygosity analysis on microdissected tissue (Tsuda et al., 2004).
DOP-PCR uses Taq polymerase and semi-degenerate oligonucleotides (CGACTCGAGNNNNNNATGTGG) that bind at a low annealing temperature at approximately one million sites in the human genome. The first cycles are followed by a large number of cycles with a higher annealing temperature, allowing only for the amplification of the fragments that were tagged in the first step. DOP-PCR generates, like PEP, fragments that are in average 400-500 bp, with a maximum size of 3 kb, although Kittler and coworkers reported a DOP-PCR method that was able to produce fragments up to 10 kb. On the other hand, a low input of genomic DNA (less than 1 ng) decreases the fidelity and the genome coverage and increases the likelihood of ADO (Kittler et al., 2002).
LMP, TLAD and MDA
The most recently developed WGA methods are the ligation-mediated PCR (LMP), the T7-based linear amplification of DNA (TLAD) and the multiple displacement amplification (MDA).
- LMP is an upcoming method that uses endonuclease or chemical cleavage to fragment the gDNA sample and linkers and primers for its amplification. It was first described by Ludecke and coworkers (Ludecke et al., 1989) and was later adapted for the WGA of small quantities of gDNA and single cells (Klein et al., 1999; Tanabe et al., 2003). Rubicon Genomics commercialises different kits (Omniplex) that allow for the amplification of RNA, DNA and methylated DNA sequences. The main advantages are that the method is able to amplify degraded DNA, and allows for different variations and that all steps are performed in the same tube. The main disadvantages are that it only amplifies a representation of the genome and it generates fragments only up to 2 kb. It has been reported to provide better CGH results than DOP-PCR (Pirker et al., 2004) but worse genotyping results than MDA (Bergen et al., 2005a).
- TLAD is a variant on the protocol originally designed by Phillips and Eberwine to amplify mRNA (Phillips and Eberwine, 1996) that has been adapted for WGA (Liu et al., 2003). It uses Alu I restriction endonuclease digestion and a terminal transferase to add a polyT tail on the 3’ terminus. A primer is then used with a 5’ T7 promoter and a 3’ polyA tract, and Taq polymerase is used to synthesise the second strand. Then the sample is submitted to in vitro transcription reaction and posterior reverse transcription. The major advantage is that TLAD does not introduce sequence and length-dependent biases. Up to now, it has not been widely used, probably because the protocol is cumbersome and time-consuming (Hughes et al., 2005).
- MDA is a non-PCR-based isothermal method based on the annealing of random hexamers to denatured DNA, followed by strand-displacement synthesis at constant temperature (Blanco et al., 1989). It has been applied to small genomic DNA samples, leading to the synthesis of high molecular weight DNA with limited sequence representation bias (Lizardi et al., 1998; Dean et al., 2002). As DNA is synthesized by strand displacement, a gradually increasing number of priming events occur, forming a network of hyper-branched DNA structures. The reaction can be catalysed by the Phi29 DNA polymerase or by the large fragment of the Bst DNA polymerase. The Phi29 DNA polymerase possesses a proofreading activity resulting in error rates 100 times lower than the Taq polymerase (Eckert and Kunkel, 1991; Esteban et al., 1993). Recently, it has been shown that MDA, when used on genomic DNA sequences with high variability, results in a loss of heterozygosity (Murthy et al., 2005). The technology has been shown to be very sensitive and can amplify from single cells (Hellani et al., 2004, Handyside et al., 2005) and single bacteria (Raghunathan et al., 2005).