Single Cell
Biology: A key research area
Development and
commercialization of economical, easier-to-use single cell tools have
enabled more researchers to explore this novel area
The ultimate biological unit
lies within a single cell. Many biological disciplines aim to elucidate
the causes of cellular differentiation at this level. The secret
triggers that signal human maturation, regeneration and genetic
diseases lie buried in a single cell that was originally part of the
genetically cloned, multicellular organism. Despite careful work with
sophisticated instrumentation available for the dissection of tissue
samples, several studies suggest that pooled cell samples, thought to
be homogeneous, often composed of cells with quite different
phenotypes.1
Development and commercialization of economical, easier-to-use single
cell tools have enabled more researchers to explore this novel area.
These include means to isolate single cells, such as fluorescence
activated cell sorting, laser capture microdissection, optical tweezers
and atomic force microscopy. Once single cell samples were readily
available, applications such as fluorescent in situ hybridization
(FISH)2 and single cell PCR3 could be used to identify the differences
between populations of single cells. This has resulted in an explosion
of work and speculation in the field of single-cell biology.4-6
Adapting multicell/tissue techniques to single cell study often has
limited utility because of technical shortcomings, with problems mostly
related to sensitivity. Despite the raw potential for single cell
genomic analysis, the field has been restricted to comparative analysis
of relatively few genomic loci for large numbers of single-cell
isolates. Techniques such as FISH or single-cell PCR can only be used
to probe a small number of DNA sequences before the cell is destroyed.
Likewise, the small sample size of a single cell has so far allowed
limited investigation of gene expression, proteomic make up and the
characterization of cell metabolites.
Whole genome amplification (WGA), a non-specific amplification
technique, offers means to overcome the above restrictions for single
cell genomic analyses. There are three different strategies for WGA
which have been observed so far.
History
Linker adapter PCR was first described in 1989.9 In this method, the
target DNA is digested with an appropriate restriction enzyme and then
each end is ligated to an adapter. These known adapter sequences are
used to uniformly amplify each of the many DNA fragments representing
the original sample. The method relies on absolutely efficient ligation
and unbiased amplification between the identical primed regions.
Primer extension preamplification (P E P) PCR, in contrast, uses a set
of random hexamers to prime template DNA.10 The subsequent thermal
cycling conditions use very low (permissive) annealing temperatures and
fifty or more cycles to create a series of fragments representing the
original input DNA. Bias in the resulting P E P PCR product is due to
nonuniformity of random hexamer annealing and extension-DNA
section with infrequent or distant priming events tend to be
discriminated in this method.
These shortfalls were largely overcome with multiple strand
displacement (MSD) amplification.11 The MSD technique employs a unique
and highly processive mesophillic DNA polymerase, phi29. The resulting
product consists of long, 1,050 kb fragments, but good amplification
and representation.
Finally, degenerative oligonucleotide primer (DOP) PCR, also described
as arbitrary PCR, relies on a set of oligos with a random
3'-end and partially fixed 5'-sequence.12 These
primers are designed to anneal DNA sample. Once extended by a
polymerase, these products are amplified using oligos by targeting
their fixed sequences. Primer design is critical for this technique
– the oligo must bind evenly throughout the DNA sequence but
not bind to other oligonucleotides. This method has also been
successfully applied to give representative samples.
Single cell analysis
Each of these techniques has been applied to the problem of amplifying
the genetic material in a single cell, and has met with some success. P
E P PCR was the first to be applied to single cell WGA, and was
successfully applied in several subsequent applications.13-14 A variant
of the DOP PCR, developed by Rubicon Genomics, was used to amplify
single chromosomes,15 a feat very shortly followed by the use of a
linker-adaptor PCR method to completely amplify a single chromosome.16
Finally, MSD with phi29 was used to amplify a series of single cells.17
WGA methods differ in two respects: the amount of bias in the product
when using limited amounts of input template and the quality
requirements for the input template. The former issue, which in single
cell applications manifests itself as apparent loss of information or
allelic drop out (ADO), is thought to be due to inequities of local
distribution of the reagents near the target.18 The latter phenomenon
is dependent on the method, and the fact that damaged DNA can make
certain loci unamplifiable.
WGA methods that generate long amplicons like MSD, can be less robust
because priming events are necessarily few and therefore any error in a
long amplicon causes a relatively large loss of information. WGA that
generates short amplicons such as P E P PCR, linker-adaptor
amplification and DOP PCR lose less information in such circumstances.
Both DOP PCR and MSD amplification are now available in commercial
molecular biology kits, some of which have been developed specifically
for single cell applications. At the heart of this product line is a
PCR-based WGA method that employs degenerate oligonucleotides coupled
with universal adaptors in a combination of P E P and DOP amplification
methods.
One commercially available single cell WGA kit can produce a
million-fold amplification of a flow-sorted or laser microcaptured
single cell resulting in approximately 5µg of final yield.29
Advances in single cell WGA will allow researchers to uncover the
contribution of genomics to single cell biology. Specifically, cancer
and drug discovery research within genomics shows the greatest
potential. Chromosomal aberrations, as a result of cancer, could be
better cataloged when comparing a single cancerous cell to its normal
counterpart. In addition, comparing single cell from the
'treated' population to the
'untreated' to evaluate genomic effects can be used
to screen drug candidates.
Understanding differences at the level of a single cell is the ultimate
goal of biology. New, commercialized techniques, such as single cell
WGA, are opening a new frontier for further study. Considerable work
has already been accomplished towards the sensitive, unbiased
amplification of single cell RNA to allow single cell expression7,
22-27 and this area has already seen the development of commercialized
kits to respond to this customer need. As researchers continue to find
sensitive means to explore epigenetics, proteomics,18,19 metabolomics
and cell signaling,20,21 the whole world of single cell biology will be
revealed.
References:
1 Reyes F, et al., (1976) The
heterogeneity of erythrocyte antigen distribution in human normal
phenotypes: an immunoelectron microscopy study, Br J Haematol., 34(4),
61321
2 Cheung SW, et al., (1977) Gene mapping
by fluorescent in situ hybridization, Cell Biol Int Rep., 1(3), 25562
3 Mariani BD, et al., (1984) Gene
amplification in a single cell cycle in Chinese hamster ovary cells, J
Biol Chem., 259(3), 190110
4 Lange, BM, (2005) Single cell genomics,
Curr Opin Plant Biol., 8(3), 23641
5 Liu WT, et al., (2005) Environmental
microbiology on a chip and its future impacts, Trends Biotechnol.,
23(4),1749
6 Diks SH, et al., (2004) Single cell
proteomics for personalized medicine. Trends Mol Med., 10(12), 5747
7 Subkhankulova T, et al., (2006)
Comparative evaluation of linear and exponential amplification
techniques for expression profiling at the single cell level. Genome
Biol., 7(3), R18
8 Paul P, et al., (2005) Single molecule
dilution and multiple displacement amplification for molecular
haplotyping, Biotechniques, 38(4), 5539
9 Ludecke HJ, et al., (1989) Cloning
defined regions of the human genome by microdissection of banded
chromosomes and enzymatic amplification. Nature, 338(6213), 34850
10 Zhang L, et al., (1992) Whole genome
amplification from a single cell: implications for genetic analysis.
Proc Natl Acad Sci USA, 89(13), 584751
11 Dean FB, et al., (2001) Rapid
amplification of plasmid and phage DNA using Phi 29 DNA polymerase and
multiply primed rolling circle amplification. Genome Res., 11(6), 10959
12 Telenius H, et al., (1992) Degenerate
oligonucleotide primed PCR: general amplification of target DNA by a
single degenerate primer. Genomics, 13(3), 71825
13 Barrett MT, et al., (1995) Genotypic
analysis of multiple loci in somatic cells by whole genome
amplification, Nucleic Acids Res., 23(17), 3488–3492
14 Snabes MC, et al., (1995)
Preimplantation single cell analysis of multiple genetic loci by whole
genome amplification. Proc Natl Acad Sci USA, 91(13), 61815
15 Gribble S, et al., (2004) Chromosome
paints from single copies of chromosomes, Chromosome Res. 12(2), 14351
16 Thalhammer S, et al., (2004)
Generation of chromosome painting probes from single chromosomes by
laser microdissection and linker adaptor PCR, Chromosome Res. 12(4),
33743
17 Hellani A, et al., (2004) Multiple
displacement amplification on single cell and possible PGD
applications. Mol Hum Reprod., 10(11), 84752
18 Irish JM, et al., (2006) Mapping
normal and cancer cell signaling networks: towards single cell
proteomics. Nat Rev Cancer, 6(2), 14655
19 Hellmich W, et al., (2005) Single cell
manipulation, analytics, and labelfree protein detection in
microfluidic devices for systems nanobiology. Electrophoresis, 26(19),
368996
20 Irish JM, et al., (2004) Single cell
profiling of potentiated phosphoprotein networks in cancer cells. Cell,
118(2), 21728
21 Krutzik PO, et al., (2003)
Intracellular phosphoprotein staining techniques for flow cytometry:
monitoring single cell signaling events. Cytometry A., 55(2), 6170
22 Ginsberg SD, (2005) RNA amplification
strategies for small sample populations. Methods, 37(3), 22937
23 Kurimoto K, et al., (2006) An improved
single cell cDNA amplification method for efficient high-density
oligonucleotide microarray analysis. Nucleic Acids Res., 34(5), e42
24 Van Gelder RN, et al., (1990)
Amplified RNA Synthesized from Limited Quantities of Heterogeneous
cDNA. Proc Natl Acad Sci USA, 87,16631667
25 Dixon AK, et al., (2000)
GeneExpression Analysis at the Single Cell Level. Trends Pharmacol Sci,
21, 6570
26 Ginsberg SD, et al., (2004) Single
Cell Gene Expression Analysis: Implications for Neurodegenerative and
Neuropsychiatric Disorders. Neurochem Res, 29,105364
27 Levsky JM, et al., (2002) Single Cell
Gene Expression Profiling. Science, 297,83640
28 Findlay I, et al., (1995) Allelic
dropout and preferential amplification in single cells and human
blastomeres: implications for preimplantation diagnosis of sex and
cystic fibrosis. Hum Reprod., 10(6),160918
29 Brueck, C. Brown, S. Michalik, D.
Vassar-Nieto, E. Mueller and G. Davis. Single-Cell Whole-Genome
Amplification. 2007. CSHL Press. Genetic Variation: A Laboratory
Manual: 136-139.