MDAGenera: An Efficient and Accurate Simulator for Multiple Displacement Amplification
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Multiple displacement amplification
Multiple displacement amplification
DNA nanoball sequencing
genomic DNA
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Whole-genome amplification (WGA) techniques are used for non-specific amplification of low-copy number DNA, and especially for single-cell genome and transcriptome amplification. There are a number of WGA methods that have been developed over the years. One example is degenerate oligonucleotide-primed PCR (DOP-PCR), which is a very simple, fast and inexpensive WGA technique. Although DOP-PCR has been regarded as one of the pioneering methods for WGA, it only provides low genome coverage and a high allele dropout rate when compared to more modern techniques. Here we describe an improved DOP-PCR (iDOP-PCR). We have modified the classic DOP-PCR by using a new thermostable DNA polymerase (SD polymerase) with a strong strand-displacement activity and by adjustments in primers design. We compared iDOP-PCR, classic DOP-PCR and the well-established PicoPlex technique for whole genome amplification of both high- and low-copy number human genomic DNA. The amplified DNA libraries were evaluated by analysis of short tandem repeat genotypes and NGS data. In summary, iDOP-PCR provided a better quality of the amplified DNA libraries compared to the other WGA methods tested, especially when low amounts of genomic DNA were used as an input material.
Multiple displacement amplification
Applications of PCR
Recombinase Polymerase Amplification
genomic DNA
Primer dimer
Hot start PCR
Taq polymerase
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Laboratory method for amplifying genomic deoxyribonucleic acid (DNA) samples aiming to generate more amounts and sufficient quantity DNA for subsequent specific analysis is named whole genome amplification (WGA). This method is only way to increase input material from few cells and limited DNA contents. While PCR-based WGA methods have been under continuous development for over a decade, shortcomings of these methods enforced many researchers to switch to the use of non-PCR-based linear amplification techniques. Moreover, application of high fidelity and high possessive DNA polymerases enabled development of an isothermal WGA technique named multiple displacement amplification (MDA). MDA is not based on PCR and doses not require thermal cycling. It should be noted that, while MDA-based techniques proposed aiming to overcome the drawbacks of PCR-based methods however, MDA is still facing some challenges. It seems that PCR-based WGA methods also have some merits. One of the problems which encountered both MDA and PCR-based methods is in the amplification of degraded DNA templates. WGA methods such as T7-based linear amplification of DNA (TLAD), balanced-PCR amplification and restriction and circularization-aided rolling circle amplification (RCA-RCA) have been suggested to aim at amplification of such DNA templates. Keywords: Whole genome amplification, multiple displacement amplification (MDA), non PCR-based methods
Multiple displacement amplification
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Abstract: We describe a method for rapidly amplifying whole genomes via a Phi29 DNA polymerase‐mediated strand displacement reaction (SDR). Genomic amplification products derived from the SDR reaction resulted in high quantities of DNA suitable for polymerase chain reaction (PCR) amplification and sequencing of mitochondrial genomes. Control region sequences of DNA derived directly from PCR amplicons of extracted DNA were identical to those derived from PCR amplification of SDR genomic DNA. Effective SDR amplification and subsequent sequencing was successful across tissues sources ranging in age from 1 year to 19 years. Strand replacement reaction genomic amplification offers a means of obtaining large quantities of DNA from small amounts of tissue.
Multiple displacement amplification
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genomic DNA
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DNA nanoball sequencing
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Multiple displacement amplification (MDA) is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples) before whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA) technique where partitioning of the template DNA into thousands of sub-nanoliter droplets, each containing a small number of DNA fragments, greatly reduces the competition among DNA fragments for primers and polymerase thereby greatly reducing amplification bias. Consequently, the ddMDA approach enabled a more uniform coverage of amplification over the entire length of the genome, with significantly lower bias and non-specific amplification than conventional MDA. For a sample containing 0.1 pg/μL of E. coli DNA (equivalent of ~3/1000 of an E. coli genome per droplet), ddMDA achieves a 65-fold increase in coverage in de novo assembly, and more than 20-fold increase in specificity (percentage of reads mapping to E. coli) compared to the conventional tube MDA. ddMDA offers a powerful method useful for many applications including medical diagnostics, forensics, and environmental microbiology.
Multiple displacement amplification
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Abstract Whole-genome amplification (WGA) techniques are used for non-specific amplification of low-copy number DNA, and especially for single-cell genome and transcriptome amplification. There are a number of WGA methods that have been developed over the years. One example is degenerate oligonucleotide-primed PCR (DOP-PCR), which is a very simple, fast and inexpensive WGA technique. Although DOP-PCR has been regarded as one of the pioneering methods for WGA, it only provides low genome coverage and a high allele dropout rate when compared to more modern techniques. Here we describe an improved DOP-PCR (iDOP-PCR). We have modified the classic DOP-PCR by using a new thermostable DNA polymerase (SD polymerase) with a strong strand-displacement activity and by adjustments in primers design. We compared iDOP-PCR, classic DOP-PCR and the well-established PicoPlex technique for whole genome amplification of both high- and low-copy number human genomic DNA. The amplified DNA libraries were evaluated by analysis of short tandem repeat genotypes and NGS data. In summary, iDOP-PCR provided a better quality of the amplified DNA libraries compared to the other WGA methods tested, especially when low amounts of genomic DNA were used as an input material.
Multiple displacement amplification
Applications of PCR
Recombinase Polymerase Amplification
genomic DNA
Hot start PCR
Primer dimer
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Single cell genomics is a powerful and increasingly popular tool for studying the genetic make-up of uncultured microbes. A key challenge for successful single cell sequencing and analysis is the removal of exogenous DNA from whole genome amplification reagents. We found that UV irradiation of the multiple displacement amplification (MDA) reagents, including the Phi29 polymerase and random hexamer primers, effectively eliminates the amplification of contaminating DNA. The methodology is quick, simple, and highly effective, thus significantly improving whole genome amplification from single cells.
Multiple displacement amplification
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Whole genome amplification (WGA) is essential for obtaining genome sequences from single bacterial cells because the quantity of template DNA contained in a single cell is very low. Multiple displacement amplification (MDA), using Phi29 DNA polymerase and random primers, is the most widely used method for single-cell WGA. However, single-cell MDA usually results in uneven genome coverage because of amplification bias, background amplification of contaminating DNA, and formation of chimeras by linking of non-contiguous chromosomal regions. Here, we present a novel MDA method, termed droplet MDA, that minimizes amplification bias and amplification of contaminants by using picoliter-sized droplets for compartmentalized WGA reactions. Extracted DNA fragments from a lysed cell in MDA mixture are divided into 105 droplets (67 pL) within minutes via flow through simple microfluidic channels. Compartmentalized genome fragments can be individually amplified in these droplets without the risk of encounter with reagent-borne or environmental contaminants. Following quality assessment of WGA products from single Escherichia coli cells, we showed that droplet MDA minimized unexpected amplification and improved the percentage of genome recovery from 59% to 89%. Our results demonstrate that microfluidic-generated droplets show potential as an efficient tool for effective amplification of low-input DNA for single-cell genomics and greatly reduce the cost and labor investment required for determination of nearly complete genome sequences of uncultured bacteria from environmental samples.
Multiple displacement amplification
genomic DNA
Single-Cell Analysis
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Multiple displacement amplification
STR analysis
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Large amounts of DNA are frequently required for use in detection assays and genomic analysis. The limited availability of DNA can be a critical obstacle to meeting research and clinical needs. DNA amplification methods are often required to generate sufficient material from small specimens or environmental samples with low DNA content. The MDA (multiple displacement amplification) reaction is increasingly the method of choice for many applications because of its extensive coverage of the genome, the generation of extremely long DNA products compared with older whole genome amplification methods and the high DNA yields, even from exceedingly low amounts of starting material. Remarkably, MDA enables genomic sequencing even from single microbial cells. Some of the uses of MDA and its strengths and limitations will be discussed.
Multiple displacement amplification
genomic DNA
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