Page 671 - Clinical Hematology_ Theory _ Procedures ( PDFDrive )
P. 671
CHAPTER 31 ■ Molecular Diagnostic Techniques and Applications 655
In laboratories in which PCR is per ormed requently, any
MOLECULAR GENETICS IN HEMATOLOGY
alse-positives are generally caused by amplicon contamina-
echniques in molecular genetics are beginning to be used tion. A broken capillary tube or a PCR plate le carelessly at
extensively in hematology. A wide range o abnormalities the edge o a table can aerosolize those amplicons, which can
can be detected with these techniques. PCR is an in vitro then adhere to lab coats and objects in the room.
method that ampli es low levels o speci c DNA sequences A simple and e ective way to combat amplicon contami-
in a sample to higher quantities suitable or urther analysis. nation is to wipe down everything—equipment, worksta-
PCR analysis can lead to the detection o gene mutations that tions, and pipettes—with bleach. Generously spray with 10%
signi y the early development o cancer. bleach and then let it sit or 15 to 30 minutes.
Microarrays (DNA chips) are basically the product o Polym erase Chain Reaction
bonding or direct synthesis o numerous speci c DNA probes
on a stationary, o en silicon-based, support. Molecular biol- PCR is an in vitro method that ampli es low levels o speci c
ogy provides new ways to establish a diagnosis, determine DNA sequences in a sample to higher quantities suitable or
patient prognosis, and monitor. urther analysis. T e three important applications o PCR are
1. Ampli cation o DNA
Single Nucleotide Polymorphisms 2. Identi cation o a target sequence
Single nucleotide polymorphisms (SNPs) comprise the most 3. Synthesis o a labeled antisense probe
abundant source o genetic variation in the human genome. PCR is unrivaled as a means or direct cloning and gene
Since the decoding o the human genome and the result- sequence analysis. T e rst diagnostic application o PCR tech-
ing greater than 3 million SNPs, laboratory techniques have nology was in prenatal diagnosis o sickle cell anemia through
been able to associate disease states and pharmacological ampli cation o beta globin sequences. PCR has become
responses with individual SNPs. SNPS have various charac- increasingly popular or detecting chromosomal breakpoints,
teristics (Box 31.1). usion genes, and MRD a er chemotherapy or leukemia and
lymphoma. However, PCR does have limitations (Box 31.2).
Polymerase Chain Reaction o use this technology, the target sequence to be ampli ed
must be known. ypically, a target sequence ranges rom
Am plicons and Am plicon Control Measures
An amplicon is a piece o genetic material, such as DNA, that
can be ormed as the product o a natural event or arti cial
ampli cation technique, such as a polymerase chain reaction BOX 31.2
(PCR). A molecular diagnostic laboratory that per orms in
vitro ampli cation reactions needs to practice techniques to Limitations and Potential Problems w ith PCR
control contamination. T is is especially true i a high num-
ber o thermal cycles is used or the PCR. Weaknesses o PCR technique include the ollowing:
PCR is highly sensitive, but a disadvantage to the use o
this assay is that it is prone to producing alse-positive results. ■ Contamination
■ Large deletions in sequence result in no place or a
primer to bind
■ Must know that PCR can ampli y across rom a large
BOX 31.1 deletion rom an adjacent site
■ Should not use PCR or mutation search unless it is
Single Nucleotide Polymorphisms known that the mutation does not involve large deletions
Potential problems with PCR include the ollowing:
■ Are not completely synonymous with point mutation
■ O en cause a premature stop codon or a missense ■ Assumes 100% e ciency o replication with each cycle
codon ■ Ampli cation in the second phase may not be truly
■ Are the most common clinically signi icant DNA exponential
polymorphism ■ A variety o variables cannot be ully controlled
■ Can be studies by allele-speci c PCR, melt curve analy- ■ Te number o cycles is di cult to determine because
sis, and microarrays o the rapidity o exponential ampli cation
■ Occur at speci c regions in the genome ■ Very low amounts o starting material may ail to
■ Can vary between populations or ethnic groups ampli y
From Heriot K. Welcome to the beginning: molecular pathology or the From Heriot K. Welcome to the beginning: molecular pathology or
community hospital pathologist and medical technologist, ASCP Annual the community hospital pathologist and medical technologist, ASCP
Meeting, ampa, FL, 2014. Annual Meeting, ampa, FL, 2014.
