Recent advances and application in whole-genome multiple displacement amplification

Background:The extremely small amount of DNA in a cell makes it difficult to study the whole genome of single cells,so whole-genome amplification(WGA)is necessary to increase the DNA amount and enable downstream analyses.Multiple displacement amplification(MDA)is the most widely used WGA technique.Results:Compared with amplification methods based on PCR and other methods,MDA renders high-quality DNA products and better genome coverage by using phi29 DNA polymerase.Moreover,recently developed advanced MDA technologies such as microreactor MDA,emulsion MDA,and micro-channel MDA have improved amplification uniformity.Additionally,the development of other novel methods such as TruePrime WGA allows for amplification without primers.Conclusion:Here,we reviewed a selection of recently developed MDA methods,their advantages over other WGA methods,and improved MDA-based technologies,followed by a discussion of future perspectives.With the continuous development of MDA and the successive update of detection technologies,MDA will be applied in increasingly more fields and provide a solid foundation for scientific research.

Simple, Rapid and Sensitive Detection of Pseudomonas aeruginesa by Colorimetric Multiple Cross Displacement Amplification

Pseudomonas aeruginosa(P:aeruginosa)is a major opportunistic pathogen in hospital-acquired infections.Thus,carly diagnosis is the best strategy for fighting against these infections.In this report,we incorporated multiple crOsS displacement amplification(MCDA)combined with the malachite green(MG)for rapid,sensitive,specific and visual detection of P.aeruginosa by targeting the oprl gene.The MCDA-MG assay was conducted at 67°C for only 40 min during the amplification stage,and then products were directly detected by using colorimetric indicators(MG),eliminating the use of an electrophoresis instrument or amplicon analysis equipment.The entire process,including specimen processing(35 min),amplification(40 min)and detection(5 min),can be finished within 80 min.All 30 non-P.aeruginosa strains tested negative,indicating the high specificity of the MCDA primers.The analytical sensitivity of the MCDA-MG assay was 100 fg of genomic templates per reaction in pure culture,which was in complete accordance with MCDA by gel clectrophoresis and real-time turbidity.The assay was also successfully applied to detecting P.aeruginosa in stool samples.Therefore,the rapidity,simplicity,and nearly equipment free platform of the MCDA-MG technique make it possible for clinical diagnosis,and more.

Review:Whole genome amplification in preimplantation genetic diagnosis

Preimplantation genetic diagnosis (PGD) refers to a procedure for genetically analyzing embryos prior to implantation,improving the chance of conception for patients at high risk of transmitting specific inherited disorders.This method has been widely used for a large number of genetic disorders since the first successful application in the early 1990s.Polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH) are the two main methods in PGD,but there are some inevitable shortcomings limiting the scope of genetic diagnosis.Fortunately,different whole genome amplification (WGA) techniques have been developed to overcome these problems.Sufficient DNA can be amplified and multiple tasks which need abundant DNA can be performed.Moreover,WGA products can be analyzed as a template for multi-loci and multi-gene during the subsequent DNA analysis.In this review,we will focus on the currently available WGA techniques and their applications,as well as the new technical trends from WGA products.

Measurement of copy number variation in single cancer cells using rapid-emulsification digital droplet MDA

Uniform amplification of low-input DNA is important for applications across biology,including single-cell genomics,forensic science,and microbial and viral sequencing.However,the requisite biochemical amplification methods are prone to bias,skewing sequence proportions and obscuring signals relating to copy number.Digital droplet multiple displacement amplification enables uniform amplification but requires expert knowledge of microfluidics to generate monodisperse emulsions.In addition,existing microfluidic methods are tedious and labor intensive for preparing many samples.Here,we introduce rapid-emulsification multiple displacement amplification,a method to generate monodisperse droplets with a hand-held syringe and hierarchical droplet splitter.Although conventional microfluidic devices require >10 min to emulsify a sample,our system requires tens of seconds and yields data of equivalent quality.We demonstrate the approach by using it to accurately measure copy number variation(CNV)in single cancer cells.

Antarctic thermolabile uracil-DNA-glycosylase- supplemented multiple cross displacement amplification using a label-based nanoparticle lateral flow biosensor for the simultaneous detection of nucleic acid sequences and elimination of carryover contamination

Here, we report a novel and universal methodology,termed ＂ntarctic thermolabile uracil-DNA-glycosylase （AUDG）-supplemented nucleic acid amplification techniques （NAAs） using a labeled-based nanoparticle lateral flow biosensor （LFB）＂ （AUDG-NAAs-LFB）, which merges enzymatic （AUDG） digestion of contaminant amplicons with different nucleic acid amplification techniques （NAAs）, and uses a lateral flow biosensor （LFB） for the rapid and visual confirmation of the presence of a target nucleic acid sequence. AUDG-NNAs-LFB is a one-pot, closedvessel assay, that can effectively eliminate false-positive signals arising from either carryover contaminants or the interaction between labeled primers. A new LFB was devised for detecting three targets （two amplicons generated from amplification of target sequences, and a chromatography control）, without the need for probe- hybridization or additional incubation steps. As a proof of concept, multiple cross displacement amplification （MCDA）, which is a specific, sensitive, and rapid isothermal amplification method, was selected as the model amplification technique to demonstrate the feasibility of AUDG-NAAs-LFB. As a result, we demonstrate the applicability of the AUDG-MCDA-LFB method for simultaneously detecting high-risk human papillomaviruses genotypes 16 and 18, which are the most and second-most prevalent strains of the virus reported in women worldwide. We also confirm the principle behind the AUDG-MCDA- LFB assay and validate its sensitivity, reproducibility, and specificity using serial dilutions of the type-specific plasmids, as well as clinical samples. This proof- of-concept method （AUDG-MCDA-LFB） can be easily reconfigured to detect various nudeic acid sequences by redesigning the specific MCDA primers.