详细描述：
全基因组扩增（WGA）是用于从少量样本中扩增出大量DNA的PCR技术。与常规PCR不同，WGA旨在扩增生物体的整个基因组而不是特定区域。扩增得到的基因组DNA可用于一系列下游应用，包括KASP基因分型。
那么，LGC是如何做WGA的？
目前存在两种做WGA的主要技术：
1. 使用热稳定性DNA聚合酶（PEP-PCR）进行引物延伸预扩增
2. 使用Phi29 DNA聚合酶通过链置换进行扩增
在评估了这两种技术的扩增效率、DNA对下游应用的适用性和成本效益后，我们强烈推荐种技术；引物延伸预扩增（PEP-PCR），并且LGC已经开发了基于该方法的WGA方案。该方案采用能够结合整个基因组的寡核苷酸作为引物，KlearTaq TM热启动DNA聚合酶复制基因组DNA。
我们开发了多种规格的WGA试剂盒，同时还提供WGA实验服务。WGA试剂盒设计的反应体系是25微升，试剂盒里以不同颜色的管子装着进行该实验所需要的所有组分。
Whole Genome Amplification (WGA) is a PCR technique that is used to produce large quantities of DNA from a small amount of starting material. Unlike conventional PCR, WGA is aimed at amplifying the entire genome of an organism rather than a specific region. The resultant amplified genomic DNA can then be used in a range of applications including KASP genotyping.
How do we do WGA?
Two main techniques exist for performing WGA:
1. Primer extension pre-amplification using thermostable DNA polymerases (PEP-PCR)
2. Amplification by strand displacement using Phi29 DNA polymerase.
We have evaluated both of these techniques to assess amplification efficiency, suitability of DNA for downstream applications, and cost effectiveness. We strongly favour the first technique, primer extension pre-amplification (PEP-PCR), and have developed a WGA protocol that is based on this method. This protocol employs the use of an oligonucleotide that binds throughout the genome to act as a primer, allowing replication of the genomic DNA by KlearTaq™ hot-start DNA polymerase.
We have developed a WGA kit that is available to purchase in a range of reaction sizes, and we also offer WGA as a service in our laboratories. The kits are designed for 25 µL reaction volumes, and contain all required reaction components in colour-coded tubes.
Requirements and performance of WGA
A minimum mass of 50 ng DNA is required per reaction and, if the DNA is of good quality, the product is usually amplified to a concentration of 500 - 1000 times that of the starting material. This resultant DNA should be sufficient to perform KASP genotyping on at least 1000 SNPs.
If the genomic DNA is from a species with a genome size considerably larger than that of human, the required starting mass of DNA will be greater. As a guideline, divide the genome size of your organism by the size of the human genome (3000 Mbp), and use the resulting number to multiply the amount of DNA required
e.g. Triticum aestivum (wheat): 16000 Mbp.
16000 Mbp / 3000 Mbp = 5.3
You will need 5.3 times as much wheat DNA per reaction = 50 ng x 5.3 = 265 ng.
Yields
Typically, from 30 - 50 ng of genomic DNA the WGA will yield enough DNA to perform SNP genotyping on at least 1000 SNPs. In practice if the starting DNA is good it will be in excess of this. This represents an amplification of approximately 500 - 1000 fold.
