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MALBAC Single Cell Whole Genome Amplification (WGA) Kit for Precision Medicine

Code No. Products
Size
Data Sheet
Price
YK001B MALBAC Single Cell Whole Genome Amplification (WGA) Kit
50 rxns
Protocol
inquire
YK001A MALBAC Single Cell Whole Genome Amplification (WGA) Kit
10 rxns
Protocol
inquire

MALBAC Single Cell WGA Re-Amp Kit

 

 
Nucleic acid analytical platforms (e.g. qPCR, microarray and high throughput sequencing) often require the sample to meet specific requirements for quantity and quality. Therefore, Whole genome amplification (WGA) is often required for analyzing samples with limited quantity, such as single circulating tumor cells, micro-biopsies, and single blastomere.
 
WGA methods are in general prone to amplification bias, PCR-based WGA introduces sequence-dependent bias mainly due to the exponential amplification. Multiple Displacement Amplification (MDA), which uses random priming and the strand-displacing phi29 polymerase under isothermal condition, has provided improvements over PCR-based methods but still exhibits considerable bias, again due to nonlinear amplification.
 
Single cell whole genome amplification and sequencing is highly desirable for many applications such as IVF preimplantation screening and genotyping of rare circulating tumor cells. Existing amplification methods are hindered by non-uniformity across the genome and poor reproducibility between single cell WGA replicates.
 
Single Cell Who Genome Kit is based on MALBAC (Multiple Annealing and Looping Based Amplification Cycles) technology, which carries out close-to-linear pre-amplification cycles of the entire genome using a mixture of highly-processive DNA polymerases with strand displacement activity, followed by an exponential amplification by PCR to a sufficient amount for various downstream analyses.
Fig1:Simplified diagram of the MALBAC reaction.  MALBAC Primers having a 27-nt common sequence followed by eight random nucleotides are annealed to the genomic DNA template. Strand-displacement synthesis generates partial amplicons, which are subsequently denatured from the template at 94°C. Priming to new positions on the genomic DNA template generates more partial amplicons, which increases coverage of the genome with a resulting reduction in amplification bias. Priming nad extension on the partial amplicons yield complete amplicons having the MALBAC primer sequence at 5’ end and its complementary sequence at the 3’ end. Denaturation at 94°C regenerates the original template and a now larger and more diverse pool of partial amplicoms. Full amplicons form loops, which may be resistant to subsequent amplification and hybirdization. Full amplicons are generated for eight cycles and the exponentially amplified by about 14-21 cycles using primers complementary to the common region of the MALBAC primers.
Highlight of MALBAC technology
 The MALBAC technology has improved and optimized by Yikon Genomics INC
Amplified single cell genome to ug level for about millionfold, and whole amplification procedure only need about 4hrs.
Best amplification uniformity among other similar products, can be applied for CNV & SNV Analysis
  >90% locus can be successfully amplified, locus allele drop out <10%
The amplification products can be applied for Genome Clone, qPCR, Microarray, NGS etc.
Single-Nucleotide Mismatch rate~10-5
Fig2: Uniformity Comparison on Single Cell Amplification
Fig3: Identify Rare SNVs by Single Cell Sequencing
Fig4: Detection of CNVs in Single Cells
Fig5: Reproducible locus representation in replicate single cell WGA reactions (tested 24 loci by qPCR)

 


Single cell whole genome amplification kit with Improved MALBAC WGA Technology
Based on the novel patented MALBAC technology, provides rapid, reliable and reproducible whole genome amplification  from single cells or equivalent DNA amount to produce 2-4 micrograms of amplified DNA in about four hours. The kit can also be used when the input amount of DNA is low (e.g. hundreds of cells or sub-ng DNA). With most cell types such as single blastomere, polar bodies, single cells from culture lines, single sperm, the kit is able to achieve >90% amplification success rate, and achieves consistent amplification efficiency in both AT- and GC-rich regions. The kit generates reproducible loci representation, achieving >0.9 correlation coefficient for qPCR Ct values from replicate single cell reactions. More than 90% of the loci can be amplified and analyzed by various platforms, such as real-time PCR (qPCR) and next-generation sequencing.

 

Product Features

  • A typical yield of 2-4μg amplified DNA from single mammalian cells
  • Single-tube, 3-step, 4-hr process, no intermediate purifications
  • >95% amplification success rate with flow sorted cells, or >0.5 pg of genome DNA
  • Achieves reproducible locus representation and consistent amplification efficiency in both AT- and GC-rich regions
  • <10% locus drop-out, <10% allele drop-out
Downstream Applications
Detection of mutations  SNP genotyping
CNV profiling and aneuploidy detection Whole genome and exome sequencing
Real-Time PCR Molecular cloning
Microarray CGH    
Source Material and Research Area
Biomarker discovery (CNVs, SNVs) IVF pre-implantation genetic screening
Genotyping of transgenic animals  Embryo and stem cell research
Single sperm genotyping    

 

Somatic variation  Tumor development and evolution
Cancer stem cells Circulating tumor cells

Bacteria

 Metagenomics research Microorganism genotypin

References:

1 C. Zong*, S. Lu*, A.R. Chapman*, X.S. Xie. Genome-Wide Detection of Single Nucleotide and Copy Number Variations of a Single Human Cell. Science.338, 1622-1626(2012)
2 S. Lu*, C. Zong*, W.Fan*, M. Yang*, et al.,Probing Meiotic Recombination and Aneuploidy of Single Sperm Cells by Whole Genome Sequencing using MALBAC.Science.338,1627-1630(2012)