Single cells

© Pascal Goetgheluck / Photo Researchers, Inc.

User:
Norma Nowak, Roswell Park Cancer Institute, Buffalo, NY

Project:
Conducting pre-implantation genetic diagnoses (PGD) on blastomere-derived cells.

Problem:
Cells harvested for PGD are traditionally put into a generic buffer and frozen. That treatment often results in "allele dropout," in which many chromosomal regions are not amenable to amplification.

Solution:
"You've got only one shot if you do PCR directly from single cells," says Nowak. So she puts cells right into a lysis buffer containing proteinase K immediately after isolating them. The buffer immediately begins to digest nucleases and other proteins, protecting and allowing easier access to the DNA.

Nowak uses the buffer that comes with Sigma's GenomePlex® Whole Genome Amplification Kit. She uses the kit to amplify the DNA, prior to performing PCR. This kills two birds with one stone, she says. By turning a single copy of the genome into thousands of copies, there's a better chance that the polymerases can find their targets, and there's also "a much more abundant source of DNA to do a battery of tests on" if the need should arise. "You can still lose regions of the genome," she adds, but that tends to be minimized "if the cell is procured and processed in a fashion that maximizes the integrity of the DNA."

Cost:
A 10-reaction GenomePlex WGA kit retails for $125 (Sigma-Aldrich).