where to buy generic provera online no prescription
Multiple cross displacement amplification (MCDA) is a nucleic acid amplification method commonly used for single-cell whole-genome sequencing.
Image Credit: https://www.researchgate.net/figure/The-principle-of-multiple-cross-displacement-amplification-The-schematic-showing-the_fig2_279965601
What is multiple cross displacement amplification?
Multiple cross displacement amplification (MCDA) method uses random primers and DNA polymerase to amplify the circular DNA template. The DNA can be amplified over 10,000-fold within a few hours. In contrast to polymerase chain reaction (PCR)-based methods for whole genome amplification, such as degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), MCDA covers the whole genome.
However, because of the rapid amplification process, MCDA can introduce sequence-dependent amplification bias, which is not reproducible throughout the genome from cell to cell.
MCDA is used widely to amplify low amounts of DNA templates in forensic sciences. The method can even amplify DNA samples that are damaged or contain stochastic variations, or mixed DNA samples. Also, the method can be used to detect pathogenic agents with high specificity and sensitivity.
How to perform MCDA?
The method is performed using random primers and DNA polymerase of the phage phi29, which has an inherent 3′ → 5′ proofreading exonuclease activity. This polymerase is associated with high processivity, ventolin jittery high fidelity, and exceptional strand displacement function.
In isothermal MCDA, which is a multiple strand displacement amplification method of DNA sequences, two primer sets (right set and left set) are used. The right set primers are complementary to one target DNA strand, and the left set primers are complementary to the opposite DNA strand. Upon hybridization to the target DNA sequence, the 5’ ends of primers remain distal to the DNA sequence.
One unique feature of this method is the displacement of primers during polymerase-mediated replication of the target DNA sequence. The displacement of primers by polymerase makes the newly replicated strands available for hybridization to the primers.
For whole-genome amplification using isothermal MCDA, a random set of primers is used to randomly replicate a portion of genomic DNA. As a result, many overlapping copies of the whole genome can be produced in a short period.
To perform MCDA, the DNA template is firstly denatured using heat (95°C) or chemical treatment. For amplification, random primers bind to denatured DNA, followed by DNA synthesis using phi29 DNA polymerase. Particularly, a total of 10 primers (displacement primers: 2; core primers: 2; amplification primers: 6) are used to recognize and amplify different portions of the target DNA sequence. The isothermal condition (60 – 65°C) required for MCDA can be maintained by simply using a water bath or a heater.
Detection of amplification products of MCDA is carried out by nanoparticle-based lateral flow biosensors. Lateral flow biosensors are portable, paper-based devices wherein different types of nanoparticles are used as labels for rapid, robust, and visual detection of specific DNA fragments. Amplification products labeled with fluorescein isothiocyanate and biotin can be detected by lateral flow biosensors within 2 minutes.
What are multiple annealing and looping–based amplification cycles (MALBAC)?
This method is a combination of PCR-based whole genome amplification and multiple displacement amplification wherein random primers containing common sequence tags are used. The primers are hybridized to the target DNA sequence, followed by isothermal strand displacement reaction-based amplification of the target sequence using Bst DNA polymerase (Bacillus stearothermophilus).
Because the primers contain common sequence tags, the newly formed DNA copies also contain common sequences at the ends. This leads to the formation of closed-loop amplicons and the prevention of repeated amplification. Although this method is associated with significantly less amplification bias, amplification of DNA sequences with secondary structures (hairpin loops) cannot be possible with this method. Also, DNA polymerase used in this method has low fidelity, which increases the risk of false-positive results.
What are the advantages of MCDA?
Compared to conventional PCR-based DNA amplification methods, MCDA comes with many advantages. For example, no specific information about the target DNA sequence is required to perform MCDA.
Random hexamer primers used in the method can target and amplify any possible DNA sequence. Because phi29 DNA polymerase does not dissociate easily, MCDA-generated amplification products are generally larger than that generated by PCR-based methods.
Moreover, based on the ability of phi29 DNA polymerase to use double-stranded circular DNA as a template, a multiply-primed rolling circle amplification method has been developed, which is considered to be the most robust technique for amplifying circular DNA templates with varied size.
Because of these advantages, the MCDA method is widely used for circular viral genome detection, nonculturable virus genome sequencing, circular plasmid detection, whole-genome sequencing, single nucleotide polymorphism genotyping, and new metagenome analysis.
Recently, circular plasmid and genomic DNA amplification efficiency of MCDA has been improved greatly by fusing the C-terminal domain of phi29 DNA polymerase with DNA binding domains.
- Science Direct. 2015. Multiple displacement amplification. www.sciencedirect.com/…/multiple-displacement-amplification
- Zhao F. 2019. Establishment and Application of Multiple Cross Displacement Amplification Coupled With Lateral Flow Biosensor (MCDA-LFB) for Visual and Rapid Detection of Candida albicans in Clinical Samples. Frontiers in Cellular and Infection Microbiology.
- Li S. 2020. Establishment and application of a multiple cross displacement amplification combined with nanoparticles-based biosensor method for the detection of Bordetella pertussis. BMC Microbiology.
Last Updated: Oct 16, 2020
Dr. Sanchari Sinha Dutta
Dr. Sanchari Sinha Dutta is a science communicator who believes in spreading the power of science in every corner of the world. She has a Bachelor of Science (B.Sc.) degree and a Master's of Science (M.Sc.) in biology and human physiology. Following her Master's degree, Sanchari went on to study a Ph.D. in human physiology. She has authored more than 10 original research articles, all of which have been published in world renowned international journals.
Source: Read Full Article