MALBAC

Multiple Annealing and Looping Based Amplification Cycles (a.k.a. MALBAC) is a quasilinear whole genome amplification method. Unlike conventional DNA amplification methods that are non-linear or exponential (in each cycle, DNA copied can serve as template for subsequent cycles), MALBAC utilizes special primers that allow amplicons to have complementary ends and therefore to loop, preventing DNA from being copied exponentially. This results in amplification of only the original genomic DNA and therefore reduces amplification bias. MALBAC is “used to create overlapped shotgun amplicons covering most of the genome”. For next generation sequencing, MALBAC is followed by regular PCR which is used to further amplify amplicons.

Prior to MALBAC, a single cell is isolated by various methods including laser capture microdissection, microfluidic devices, flow cytometry, or micro pipetting, then lysed. MALBAC single-cell whole-genome amplification involves 5 cycles of quenching, extending, melting, and looping.

The major advantage of MALBAC is that DNA is amplified almost linearly. The utilization of specialized primers enables looping of amplicons which then prevents them from being further amplified in subsequent cycles of MALBAC. These primers are 35 nucleotides long, with 8 variable nucleotides that hybridize to the templates and 27 common nucleotides. The common nucleotide sequence is GTG AGT GAT GGT TGA GGT AGT GTG GAG. The 8 variable nucleotides anneal randomly to the single stranded genomic DNA molecule. After one extension, semi-amplicon, an amplicon containing the common nucleotide sequence on only the 5’ end, is made. This semi-amplicon is used as a template for another round of extension, which then results in a full-amplicon, an amplicon where the 3’ end is complementary to the sequence on the 5’ end.

MALBAC primers have variable components which allow them to randomly bind to the template DNA. This means that on a single fragment at any cycle, there could be multiple primers annealed to the fragment. A DNA polymerase such as one derived from Bacillus stearothermophilus (Bst polymerase) is able to displace the 5’ end of another upstream strand growing in the same direction.

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