section 23.7

Diagnostic and Clinical Applications of DNA

535

TABLE 23-2

A Partial List o f Diseases for Which Single Genes Have Been Isolated and Characterized*

Familial amyloid polyneuropathies

Niemann-Pick disease

Phenylketonuria

X-linked Charcot-Marie-Tooth disease

Familial hypercholesterolemia

Familial colon cancer

Christmas factor deficiency

Hereditary retinal degenerative diseases

Retinoblastoma, Wilson’s disease

Ototoxic deafness

Type IV collagen deficiencies

XSCID

Hydatidiform moles and choriocarcinomas

Compulsive disorders

Tay-Sachs disease

Myotonic muscular dystrophy

Alzheimer’s disease

Familial dysalbuminemic hyperthuroxinemia

Gaucher’s disease

Long QT syndrome

Charcot-Marie-Tooth 1A disease

Cerebral autosomal dominant arteriopathy

Williams syndrome

Cancer-associated mar binding protein

Von Hippel-Lindau disease

Breast or ovarian cancer

Breast cancer (BRCA1)

Idiopathic dilated cardiomyopathy

Prostate cancer

Hereditary neuropathy with liability

*Each of these mutant alleles contributes to a greater or lesser degree to the pathology of the disease or inherited disorder. As of December

1997, patents had been issued to universities or companies for the commercial use of these genes.

Forensic DNA Analysis

RFLPs

are also widely used in forensic pathology,

criminology, and cases of contested paternity. A par-

ticular set of DNA probes is specific for hypervari-

able sequences in the human genome that are inher-

ited in a Mendelian pattern identical to the inheritance

of genes.

Hypervariable sequences

are highly polymor-

phic minisatellite loci that are unique to each individ-

ual just as each individual has a unique set of genes.

These hypervariable sequences can be detected by a

special set of DNA probes called

Jeffreys probe

af-

ter their discoverer. A Southern blot analysis of frag-

ments of DNA from an individual using the Jeffreys

probe constitutes a unique “genetic fingerprint.” Only

monozygous twins have the same pattern of hyper-

variable sequences and identical genetic fingerprints

(Figure 23-15).

DNA obtained from a blood stain, a human hair cell, or

even a few sperm provides enough material to match with

the DNA obtained from a suspect. DNA testing of suspects

in rape, murder, and other crimes in which a sample of

DNA was obtained at the scene of the crime is now a

routine procedure.

DNA sequences can be amplified by the

polymerase

chain reaction (PCR)

technique. Only an infinitesimal

amount of DNA is needed, e.g., a sample of DNA from a

single hair follicle or sperm. The DNA to be amplified is

mixed with three other components in an automated PCR

procedure:

1. A heat-stable DNA polymerase that is isolated from a

thermophilic bacterium,

2. An excess of two short primer DNAs that are

complementary to opposite strands of the DNA

fragment that is to be amplified, and

3. An excess of deoxyribonucleotide triphosphates.

PCR consists of repeated cycling of three reactions:

dénaturation of the DNA by heating, reannealing of the

primers with the target DNA by cooling, and synthesis of

new DNA strands. The sequence of reactions is automati-

cally repeated at defined intervals to yield an exponential

increase in the amount of DNA. Twenty cycles of PCR

amplify DNA by about a factor of 10

6

and 30 cycles by

about

1 0

9.

PCR amplification of DNA is one of the most widely

used techniques in medical research and diagnostics. PCR

is used in forensic pathology (to identify human remains),

in rapid identification of infectious microorganisms, in di-

agnosis of inherited diseases, and in archaeology and an-

thropology where small DNA samples can be recovered.

Sequencing DNA

Until the development of automated DNA sequencing ma-

chines in the 1990s, two techniques were used to sequence

the bases in a segment of DNA. Each of the techniques in-

volves the isolation of a restriction fragment containing

a few hundred or a few thousand base pairs. The DNA

is denatured and each strand is sequenced separately so