Introduction to Whole Genome Amplification Technology

Researchers often encounter problems with limited or insufficient DNA samples in downstream analyses. QIAGEN's Whole Genome Amplification Technology solves this problem by amplifying genomic DNA with small samples or precious samples, including single cells. The REPLI-g Kit accurately replicates raw DNA samples without bias, and the amplified DNA can be directly applied to a wide range of genetic analyses.

What is whole gemome amplification?
As a method to increase the amount of limited DNA, genome-wide amplification technology emerged in 1992, which is particularly suitable for forensic identification and genetic disease research, as well as new technologies such as second-generation sequencing technology and CGH array (comparative genomic hybridization). Technical applications, the main problem of the latter is the limited number of DNA samples, but the analytical demand is considerable. A variety of WGA technologies have been developed in the industry, and the experimental scheme and replication accuracy between them are different.

WGA Technology - Comparison of PCR Technology and Multiple Displacement Amplification (MDA) Technology
The industry has developed a variety of WGA technologies; however, these technologies differ in their accuracy and ease of use. QIAGEN's REPLI-g Kit uses WGA technology called multiple displacement amplification to provide accurate, whole-genome amplification without bias.

PCR-based WGA technology

There are two main types of PCR-based WGA techniques: degenerate oligonucleotide PCR (DOP-PCR) technology (1) and pre-amplification primer extension (PEP) technology (2). The main difference between the two technologies is that the PEP technique uses random primers and low PCR annealing temperatures, while the DOP-PCR technique uses semi-degenerate oligonucleotides (such as CGACTCGAGNNNNNNATGTGG) and higher annealing temperatures. Taq DNA polymerase was used in both methods, limiting the length of amplification to 3 kb (average fragment length of 400–500 kb) and introducing errors into the amplified sequence. Moreover, studies have found that the genomic coverage of these techniques is not complete enough and will be biased in the amplification - due to the preferential binding of primers to certain specific regions, some sequences in the DNA amplification products are relatively increased.

Multiple displacement amplification of WGA technology

REPLI-g uses a thermostatic genomic amplification technique called multiple displacement amplification (MDA), which involves the binding of random hexamers to denatured DNA and strand displacement using Phi 29 polymerase at constant temperature. Synthesis. After the primers are added to each of the replacement strands, a branched DNA structure network is formed. Phi 29 polymerase does not dissociate from the genomic DNA template, which allows the resulting unbiased DNA fragments to be extended to 100 kb. The enzyme also has an exonuclease-correcting activity from the 3' end to the 5' end, which provides up to 1000-fold fidelity compared to Taq DNA polymerase-based methods (see PCR-based WGA technique above). Phi 29 polymerase is supported by a unique REPLI-g optimized buffer, which can easily overcome secondary structural difficulties such as hairpin loops, avoiding slippage, customization and polymerase dissociation during amplification. Such unbiased DNA fragments can be synthesized to a length of 100 kb.

Advantages of MDA technology compared to PCR-based WGA method

Traditional genomic DNA amplification methods include the time-consuming EBV transformed cell line establishment process, followed by PCR whole genome amplification steps using random or degenerate oligonucleotide primers. PCR-based WGA methods commonly used by other manufacturers also produce non-specific amplification interference and incomplete site coverage. DNA fragments shorter than 1 kb in length may be generated in some cases and are not available in many subsequent applications. In general, due to the use of low-fidelity Taq DNA polymerase, the resulting DNA contains a high rate of base mutation, thus forming an error-prone amplification, resulting in a single base pair mutation, STR. The occurrence of adverse consequences such as shortening and expansion. REPLI-g does not have these shortcomings. It can perform a highly uniform amplification of the entire genome, with minimal deviations and mutation rates during the reaction.

The application of WGA technology using REPLI-g amplified genomic DNA is suitable for a wide variety of subsequent applications, including:

• SNP genotyping using TaqMan primer/probe set
• Mutation detection based on qPCR and PCR techniques
• Second generation sequencing
• STR/microsatellite sequence analysis
• Sanger sequencing
• RFLP and Southern blot analysis
• Such as comparative genomic hybridization, chip analysis technology

The above information is from QIAGEN: http://

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