PCR

1. Dr. P. Suganya
Assistant Professor
Department of
Biotechnology
Sri Kaliswari College
(Autonomous), Sivakasi

2.  Single-cell molecular-biology is a relatively new scientific
branch in biology. The first single-cell analysis were
involved in the characterization of mitochondrial DNA in
1988. Single-cell DNA analysis, in particular genomic DNA,
is important and may be informative in the analysis of
genetics of cell clonality, genetic anticipation and single-
cell DNA polymorphisms. Nowadays for most scientists
the quantitative transcriptomics in a single-cell is much
more important, and the analytical method of choice is the
quantitative real-time RT-PCR. In single-cell biology
the absolute abundance of particular mRNAs or
microRNAs and their up- or down-regulation in a single
cell, compared to their neighbour cells, is the goal. The
need for quantitative single-cell mRNA analysis is evident
given the vast cellular heterogeneity of all tissue cells and
the inability of conventional RNA methods, like northern
blotting, RNAse protection assay or classical block RT-PCR,
to distinguish individual cellular contributions to mRNA
abundance differences.

3.  The polymerase chain reaction (PCR) has a
wealth of applications in research,
 but perhaps one of its most challenging roles
in single cell analysis, a
 mplifying the DNA from just a single cell,
 or a very small sample of cells.


4.  Real-time single cell PCR can be used in gene
expression analysis. This has a role in looking at
the differences in gene expression and
elucidating mechanisms of disease between
individual cells. Many tissues, both healthy and
diseased, are heterogeneous – for example, the
pancreas includes alpha cells, beta cells, delta
cells, PP cells and epsilon cells. Tumours can be
a mixture of different cells, and two tumours in
the same individual can have very different
populations of cells. Because of this, looking at
the gene expression profile from a number of
single cells is likely to give a more
representative view of the population.

5.  Single cell PCR can be used to compare the gene
expression profiles in cells from healthy people
and people with a certain disease, or even
looking at differences between healthy and
diseased cells in one individual, or two
populations of healthy cells in the same organ.
Gene expression analysis can also help to explore
the mechanisms behind self-renewal and
differentiation in human pluripotent stem cells.
 In an example, researchers from the Queensland
Institute of Medical Research used single cell
PCR to look at the different patterns of
expression of cytokine genes in populations of
CD4 cells following exposure to an immune
stimulus in mice.

6.  Single cell PCR is used in metagenomics
research to look at the genes in mixed
populations of bacteria and see who these
genes in individual bacteria affect the
population as a whole. Single cell PCR is
particularly important in looking at those
bacteria that are difficult to culture in the
laboratory.
 The technique also has a role in the
development of transgenic
animals developed as models for drug and
disease research.

7.  Single cell PCR is used in pre-implantation
diagnosis, where a single cell is removed
from the embryo to test for inherited
diseases such as beta-thalassaemia during IVF
(in vitro fertilisation). It also has a role in
prenatal diagnosis for genetic disorders, and
antenatal screening.
 Being able to detect rare mutants could be
useful for very early diagnosis of leukaemias
and lymphomas by spotting chromosomal
changes.

8.  PCR analysis from a single cell or from very
small samples of cells is challenging because
it involves minute quantities of DNA, and can
miss changes on non-amplified alleles (allele
drop-out).
 Identified the wilson diseases