Single-cell analysis

In the field of cellular biology, single-cell analysis is the study of: genomics, transcriptomics, proteomics and metabolomics at the single cell level. Due to the heterogeneity seen in both eukaryotic and prokaryotic cell populations, analyzing a single cell makes it possible to discover mechanisms not seen when studying a bulk population of cells. Technologies such as fluorescence-activated cell sorting (FACS) have increased the throughput of single cell sorting, and increased the development of single cell analysis techniques. The development of new technologies are increasing our ability to sequence the genome, and transcriptome, of single cells, as well as to quantify their proteome and metabolome.

The first step of single-cell analysis is the isolation of single cells. There are 7 methods currently used for single cell isolation: serial dilution, micromanipulation, laser capture microdissection, FACS, microfluidics, manual picking, and Raman tweezers.

Manual single cell picking is a method is where cells in a suspension are viewed under a microscope, and individually picked using a micropipette. Raman tweezers is a technique where  Raman spectroscopy is combined with optical tweezers, which uses a laser beam to trap, and manipulate cells.

Single-cell genomics is heavily dependent on increasing the copies of DNA found in the cell so there is enough to be sequenced. This has led to the development of strategies for whole genome amplification (WGA). One widely adopted WGA techniques is called Degenerate Oligonucleotide–Primed Polymerase Chain Reaction (DOP-PCR). This method uses the well established DNA amplification method PCR to try and amplify the entire genome using a large set of primers. Although simple, this method has been shown to have very low genome coverage. An improvement on DOP-PCR is Multiple Displacement Amplification (MDA), which uses random primers and a high fidelity enzyme, usually Φ29 DNA polymerase, to accomplish the amplification of larger fragments and greater genome coverage than DOP-PCR. Despite these improvement MDA still has a sequence dependent bias (certain parts of the genome are amplified more than others because of their sequence). The method shown to largely avoid the bias seen in DOP-PCR and MDA is Multiple Annealing and Looping–Based Amplification Cycles (MALBAC). Bias in this system is reduced by only copying off the original DNA strand instead of making copies of copies. The main draw backs to using MALBA, is it has reduced accuracy compared to DOP-PCR and MDA due to the enzyme used to copy the DNA. Once amplified using any of the above techniques, the DNA is sequenced using next-generation sequencing (NGS).

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