Page 675 - Clinical Hematology_ Theory _ Procedures ( PDFDrive )

CHAPTER 31 ■ Molecular Diagnostic Techniques and Applications 659

FIGURE 31.5 PCR with capillary elec-

trophoresis technique using three pairs
o primers or the detection o immu-

noglobulin heavy-chain gene rear-

rangements. Frameworks I and II show

a monoclonal peak, representing gene

rearrangement. Framework III shows a
polyclonal pattern. (From Sun . Flow

Cytometry, Immunohistochemistry, and

Molecular Genetics or Hematologic
Neoplasms, 2nd ed, Philadelphia, PA:

Lippincott Williams & Wilkins, 2012.)

suited or handling small amounts o DNA. T e rst pub- method is considered to be the “gold standard” method to

lished papers on DNA analysis by CE only appeared in 1988. which other molecular methods are compared. However,

Various CE methods and their applications to nucleic acid the Sanger method does have limitations (Box 31.3). DNA

analysis, speci cally those dealing with nucleosides, nucleo- sequencing displays the exact nucleotide or base sequence o

tides, oligonucleotides, and dsDNA (PCR) ragments, have a ragment o DNA that is targeted.

been developed. T e Sanger method uses a series o enzymatic reactions

In contrast to conventional gel electrophoresis, CE takes to produce segments o DNA complementary to the DNA

advantage o two types o driving orces: (1) the orce causing being sequenced. T is method is also called the chain ter-

the electrophoretic migration and (2) the orce exerted by mination method because a er synthetic nucleotides that

electroosmotic f ow (EOF) through the capillary. CE o ers lack the −OH at 3′ carbon atom are added to the growing

several similarities to high-per ormance liquid chromatog- DNA strand, there is no 3′-OH or the next nucleotide to be

raphy (HPLC), that is, ease o use, high resolution, speed, attached to, and the DNA chain stops elongating.

on-line detection, and ull automation capability. CE, hav- Automated sequencing techniques use primers with our

ing taken essential components rom both HPLC and elec- di erent f uorescent labels.

trophoresis, can be viewed as an instrumental approach to

electrophoresis. 1. T e rst step in sequencing a target is usually to ampli y it

DNA sequencing (Figs. 31.7 and 31.8) is the determina- by cloning or in vitro ampli cation, usually PCR. Once the

tion o the precise sequence o nucleotides in a sample o ampli ed DNA is puri ed rom the clinical specimen (the

DNA. T e most popular method or doing this is called the target DNA), it is heat denatured to separate the double-

dideoxy method or Sanger method. T is DNA sequencing stranded DNA (dsDNA) into single strands (ssDNA).

FIGURE 31.6 Splenic B-cell marginal

zone lymphoma. Polymerase chain

reaction with capillary electrophoresis

technique reveals a monoclonal peak in
each o the three rameworks, represent-

ing immunoglobulin heavy-chain gene

rearrangement. (From Sun . Atlas o

Hematologic Neoplasms, New York, NY:
Springer, 2009.)