Scientists can now get a more accurate look at a single cell’s genome, thanks to a new technique. Since the procedure captures more of a cell’s genetic code than methods past, researchers can better resolve small genetic changes in individual cells that drive such processes as evolution, cancer, and maybe even neurodegenerative disease. Led by Sunney Xie, researchers from Harvard University reported their method in the December 21 issue of Science. They used it to identify single nucleotide and gene copy number variations among cells in the same line. Both types of variants can cause Alzheimer's, Parkinson's, and other neurological diseases.
To generate enough DNA for sequencing, a single cell’s genome has to be amplified many times over. This can introduce errors. Some regions are easier to copy than others, for example, and can dominate a sample. This is especially true when researchers rely on the polymerase chain reaction (PCR), because it preferentially and disproportionately amplifies easily copied regions.
First author Chenghang Zong and colleagues developed a more conservative method, called multiple annealing and looping-based amplification cycles (MALBAC). It amplifies genomic DNA cautiously at first. Instead of using every DNA fragment as a template in multiple rounds of amplification, as does PCR, MALBAC loops any duplicated DNA from the first round, leaving only the original DNA as linear pieces for the next amplification step. That limits imbalances. After five cycles of MALBAC, PCR takes over to do the rest.
A new method to amplify a single cell’s DNA for sequencing reveals copy number variations (green arrows) among cells of the same line. Image courtesy of Science/AAAS
Researchers found that MALBAC covers up to 93 percent of a single human cell’s genome. With such high capture, the group easily detected copy number variations in single cells and spotted single nucleotide variations between related cells. Another key component of accuracy is haplotype phase. This tells the scientist if there are two mutations affecting both copies of a gene, or those same two mutations in only one copy, leaving the other copy unaffected. "Knowing if you have one good copy or zero is a big deal for clinical (and research) accuracy," noted George Church, Harvard Medical School, in an e-mail to Alzforum (see full comment below).—Gwyneth Dickey Zakaib
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