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152 Part IV: Molecular and Cellular Hematology Chapter 10: Genetic Principles and Molecular Biology 153

are known. If a SNP demonstrates close linkage with a disease phe- PCR can be carried out just a few nanograms of DNA, as opposed
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notype in a family or a series of families, then the disease-causing locus to the micrograms required for cloning. The amount of DNA in a several-
must be located near the SNP. This establishes the approximate location year-old blood stain, a single hair, or even the back of a licked postage
of the disease-causing locus. Linkage can be used in some cases to help stamp is often sufficient for analysis. PCR is so sensitive that under opti-
diagnose genetic disease in family members by determining whether a mal conditions the DNA from a single cell may be amplified. Moreover,
parent who has a disease-causing allele has transmitted a linked SNP the stability of DNA is such that very old preserved material may be
allele to their offspring. used. Thus, it is possible to amplify the DNA from blood films, mum-
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The Human Genome Project was initiated in 1990 and completed mies, and other ancient biologic material. In addition, because it does
in 2003. Its main goals were to discover polymorphisms useful for link- not require cloning, the procedure is much faster than older techniques.
age throughout the genome (the “gene map”) and to determine the Genetic testing for sickle cell disease, for example, can be done in a
entire human DNA sequence. 39–41 These goals have been accomplished, single day with PCR.
and DNA sequencing has become far cheaper and more efficient in Amplifying by PCR cDNA produced by reverse-transcribing
recent years (Chap. 11). Consequently, many thousands of individuals mRNA in tissue extracts (reverse transcriptase polymerase chain reac-
have now been sequenced, and the genes responsible for approximately tion [RT-PCR]) provides a very sensitive means for measuring the
4000 mendelian conditions have been identified. 14,42,43 This has greatly expression of genes in tissues. In the early cycles of PCR, the rate of
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increased our understanding of the mechanisms that underlie many amplification is a function of the amount of template; thus it allows for
diseases and has permitted more accurate genetic testing and diagnosis. quantification of mRNA or DNA. For this purpose, a housekeeping
mRNA is also measured and the ratio of this reference mRNA to tested
THE METHODS OF MOLECULAR mRNA is used. Thus, the slope of the curve can be used to measure
BIOLOGY the amount of mRNA or DNA in a specimen. This process, which has
been designated real-time PCR, has been automated by using fluores-
CLONING DNA cent probes that are destroyed during the amplification process or a dye
that binds only to double-stranded DNA.
The sequencing of DNA and the preparation of probes requires that a
fragment of DNA is amplified manyfold to provide a relatively pure sam-
ple for study. The classical method by which this is achieved, cloning, is a DNA SEQUENCING
central technique of molecular biology. It is generally accomplished by The chain termination technique has traditionally been used to deter-
3
inserting the DNA into a vector, a bacteriophage or plasmid, that nor- mine the sequence of DNA. It depends upon synthesizing a labeled
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mally replicates within a bacterial cell. When such a phage or plasmid strand of DNA, with the DNA to be sequenced serving as the template.
contains a foreign DNA fragment, the fragment also undergoes repli- The mixture of nucleotides used contains a nucleotide analogue that
cation and can then be purified in greatly amplified form. If the DNA results in chain termination when incorporated. Gel electrophoresis
is not available in pure form to begin with, it must be purified from a of the labeled products produces “ladders” of polynucleotides. The size
collection of DNA fragments that is designated a library. An adequate of each fragment depends on the point at which there exists a nucleo-
genomic library consists of millions of fragments of the genetic material tide corresponding to the chain terminating analogue in the mixture.
of a cell that have been ligated into a suitable vector. Another valuable Sequencing can be done rapidly and accurately by automated methods
type of library is made by transcribing mRNA from a tissue into com- in which the elongation of the strand is terminated by a fluorescent
plementary DNA (cDNA) using the enzyme reverse transcriptase. Such nucleotide and electrophoresis is carried out in capillary tubes rather
a cDNA library is particularly useful for the isolation of genes because than slab gels. Although DNA sequencing formerly required cloning
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in it are represented only the intron-free portions of genes that are being of the fragment to be studied, amplification by PCR serves as a simpler
actively transcribed in a tissue. In contrast, a genomic library represents alternative.
all of the genetic material, coding and noncoding, transcribed and non- The development of new high-throughput DNA sequencing technol-
transcribed. Many different vectors have been designed and they possess ogy has decreased the cost of DNA sequencing by many orders of mag-
the capacity to replicate fragments of DNA of widely differing sizes, vary- nitude (this technology is also sometimes termed “next-generation” or
ing from yeast artificial chromosomes (YACs), which may incorporate a “massively parallel” sequencing; see Chap. 11 for further details). 49–51 In
million or more base pairs of DNA, to bacterial artificial chromosomes one common approach, genomic DNA is chopped at random into small
(BACs) and P1-derived artificial chromosomes (PACs), which can con- segments, typically 100 to several hundred bp in size. Short synthetic
tain 100,000 bp, to cosmids (20,000 to 30,000 bp). Much smaller inserts, DNA sequences, termed adapters, are joined to the ends of the genomic
ranging in size from approximately 3000 to 12,000 bp can be cloned into DNA fragments. These double-stranded DNA fragments are separated
bacteriophages. into single strands and then attached to a solid surface, such as a glass
slide. Each individual DNA fragment is amplified by PCR into a cluster
THE POLYMERASE CHAIN REACTION of thousands of identical copies, using the adapters as primer sequences
Amplification of the desired part of the genome may be achieved, when (the multiple copies provide a signal strong enough to be visualized by
some of the sequence is already known, by using the polymerase chain a specilialized camera). A sequencing reaction then occurs, in which
reaction (PCR), a technique that is much simpler and more rapid than these fragment clusters serve as templates for synthesizing complemen-
cloning. For example, one may wish to determine the sequence of a tary sequences. Similar to the Sanger sequencing process described pre-
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portion of a gene for diagnostic purposes, but cloning the gene(s) of viously in the section on DNA sequencing, new complementary bases
interest is too time-consuming and labor intensive to be practical. Two (to which a base-specific fluorescent label is attached) are added one at
primers matching opposite strands of DNA on either side of the region a time. The fluorescent signal from each cluster is recorded by a camera,
of interest are used to amplify the intervening specific segment of DNA revealing the base-pair sequence of each fragment. The key advantage of
by more than a million-fold. Successive cycles of DNA synthesis from this approach is that millions of different DNA fragments are sequenced
the primers and chain separation by heating between the cycles are the simultaneously, in contrast to older methods in which only a few dozen
basis of this powerful technique. fragments are sequenced at a time.

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