Techniques of DNA analysis

With the huge increase in knowledge of the human genome
and its DNA sequence, growing numbers of disease genes can
now be examined using DNA analysis. Few laboratory tests at
the disposal of the modern clinician have the potential
specificity and information content of these techniques. Only a
few years ago, DNA analysis was mainly applicable to
presymptomatic diagnosis of inherited conditions and the
detection of carriers following initial diagnosis of the patient by
more conventional laboratory tests (e.g. biochemical and
histological). In current practice, the DNA laboratory has an
increasing role in the initial diagnosis of many diseases
by analysis of specific genes associated with mendelian
disorders.

molecular genetics laboratories

Over 20 regional molecular genetics laboratories provide a
service to the regions of the UK with many additional
laboratories providing genetic tests in areas such as
mitochondrial disease and haemoglobinopathies. The following
chapter summarises the standard techniques of DNA analysis
employed by molecular laboratories for the provision of
services to the clinician.

DNA extraction

Genomic DNA is usually isolated from EDTA-anticoagulated
whole blood, often using an automated method. In addition,
DNA can also be readily isolated from fresh or frozen tissue
samples, chorionic villus biopsies, cultured amniocytes and
lymphoblastoid cell lines. Smaller quantities of DNA can be
recovered from buccal mouthwash samples and fixed
embedded tissues, although the recovery is considerably less
reliable. The increased use of the polymerase chain reaction
(PCR) means that for a small proportion of analyses, blood
volumes of 1 ml are adequate. In many instances however,
larger volumes of blood are still required because numerous
tests are required when analysing large or multiple genes and
not all tests use PCR based methods of analysis.

Genomic DNA

Genomic DNA remains stable for many years when frozen.
This enables storage of samples for future analysis of genes that
are not yet isolated, and is crucial when organising the
collection of DNA samples for long term studies of inherited
conditions.

The polymerase chain reaction (PCR)

The use of PCR in the analysis of an inherited condition was
first demonstrated in the detection of a common -globin
mutation in 1985. Since then, PCR has become an
indispensable technique for all laboratories involved in DNA
analysis. The technique requires the DNA sequence in the gene
or region of interest to have been elucidated. This limitation is
becoming increasingly less problematic with the pending
completion of the entire human DNA sequence.

advantage of the PCR

The main advantage of the PCR method is that the regions
of the gene of interest can be amplified rapidly using very small
quantities of the original DNA sample. This feature makes the
method applicable in prenatal diagnosis using chorionic villus
or amniocentesis samples and in other situations in which
blood sampling is not appropriate.

genomic DNA

In practice, because of the way genomic DNA is organised
into coding sequences (exons) separated by non-coding
sequences (introns), analysis of even a small gene usually
involves multiple PCR amplifications. For example, the breast
cancer susceptibility gene, BRCA1, is organised into 24 exons,
with mutations potentially located in any one of them. Analysis
of BRCA1 therefore necessitates PCR amplification of each
exon to enable mutation analysis.