MALBAC – Improving the success rates of IVF

MALBAC is a fairly recent technique for whole-genome amplification such that enough DNA can be produced for sequencing. If you haven’t already I’d highly recommend reading this article on PCR written by Robb as this article builds on details described previously.

MALBAC stands for Multiple Annealing and Looping Based Amplification. It improves upon previous whole-genome amplification methods by looping the full amplicon (the replicated DNA) such that it itself cannot be amplified reducing amplification bias (where one or multiple stretches of the genome are amplified to a greater extent than others). This leads to the production of DNA fragments from across the whole genome (a higher percentage coverage) and leads to accurate sequencing of a very small amount of starting DNA. This is immensely useful in forensics or prenatal testing. It is possible to accurately detect SNPs (single-nucleotide polymorphisms) and CNVs (copy number variations) such as those present in Down’s Syndrome (duplication of chromosome 21).

How does it work?

1. At 0˚C, a mixture of random primers are allowed to anneal evenly across the genome. Each primer has 27 identical nucleotides and 8 differing nucleotides.

2. Upon subsequent addition of polymerase and increasing the temperature to 65˚C, multiple semi-amplicons of varying lengths are generated. These “semi-amplicons” are named as such because they only contain the common 27-nucleotide sequence at one end and therefore cannot circularise. Error may be introduced at this stage due to inaccurate replication, though this is tiny. These errors can be accounted for by sequencing DNA from multiple cells/sources if available.

3. These semi-amplicons are then replicated after being melted off from their template at 94˚C and the annealing of more primers. This generates full-amplicons which have complementary ends which can anneal to each other to form loops.

4. These looped full-amplicons can then sequenced. See below for a diagramatic, systematic, ultramatic representation of the MALBAC process.

MALBAC

Application in IVF

This method of amplifying the whole genome without sequence bias is particularly useful when trying to detect abnormalities within the genetic information of embryos such as chromosomal translocations which are the most common cause of miscarriages in older women.

IVF success is traditionally impaired due to the requirement for removing a cell from the developing embryo for DNA sequencing. However, by sequencing DNA from the polar bodies (the cells initially produced during embryo development), this requirement is subverted, increasing the success rates of IVF. This technique, therefore, does not detect abnormalities passed down by the father but does capture 70% of defects from the mother’s side. Currently this technique is being used with 30 women with genetic disorders and the hope is that healthy babies whose DNA has been sequenced after MALBAC amplification will be born in 2014.

Polar

The first stage of embryo development. See the polar bodies on top of the egg cell.

Happy New Year all!

James

Chenghang Zong, Sijia Lu, Alec R. Chapman and X. Sunney Xie1 (2013). Genome-Wide Detection of Single Nucleotide and Copy Number Variations of a Single Human Cell Science, 338 (6114), 1622-1626 DOI: 10.1126/science.1229164

http://www.newscientist.com/article/dn24791-genetic-test-screens-embryo-without-disturbing-it.html#.UsIP1fRdXis

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