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Molecular basis of hematology
Laura G. Schuettpelz, James R. Cook and Timothy A. Graubert
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CHAPTER
01
M olecular basis of hematology
Laura G. Schuettpelz, James R. Cook, and Timothy A. Graubert
B asic concepts, 1 Clinical applications of DNA technology Glossary, 18
A nalytic techniques, 6 in hematology, 14 Bibliography, 21
Basic concepts tide. The two strands are connected through hydrogen bonds
between strict pairs of purines and pyrimidines; that is, ade-
Advances in recombinant DNA technology over the past 25 nine must be paired with thymine (A-T) and guanine must be
years have substantially altered our view of biologic processes paired with cytosine (G-C). This is known as Watson–Crick
and have immediate relevance to our understanding of both base pairing. Consequently, the two strands of DNA are said to
normal hematopoietic cell function and hematologic pathol- be complementary, in that the sequence of one strand deter-
ogy. A complete review of molecular genetics is beyond the mines the sequence of the other through the demands of strict
scope of this chapter, but the following is intended as a review base pairing. The two strands are joined in an antiparallel
of the concepts of the molecular biology of the gene, an intro- manner so that the 59 end of one strand is joined with the 39
duction to epigenetics and genomics, an outline of noncoding end of the complementary strand. The strand containing the
RNAs, and an explanation of the terminology necessary for codons for amino acid sequences is designated as the sense
understanding the role of molecular biology in breakthrough strand, whereas the opposite strand that is transcribed into
discoveries. Emerging diagnostic and therapeutic approaches messenger RNA (mRNA) is referred to as the antisense strand.
in hematology will be reviewed. The concepts outlined in the
following sections also are illustrated in Figure 1-1; in addition,
Structure of the gene
boldface terms in the text are summarized in the glossary at the
end of this chapter. Several examples of how these concepts DNA dictates the biologic functions of the organism by the
and techniques are applied in clinical practice are included. flow of genetic information from DNA to RNA to protein.
The functional genetic unit responsible for the production of
a given protein, including the elements that control the timing
Anatomy of the gene
and the level of its expression, is termed a gene. The gene con-
Structure of DNA tains several critical components that determine both the
amino acid structure of the protein it encodes and the mecha-
DNA is a complex, double-stranded molecule composed of
nisms by which the production of that protein may be con-
nucleotides. Each nucleotide consists of a purine (adenine or
trolled. The coding sequence, which dictates protein sequence,
guanine) or pyrimidine (thymine or cytosine) base attached
is contained within exons; these stretches of DNA may be
to a deoxyribose sugar residue. Each strand of DNA is a suc-
interrupted by intervening noncoding sequences, or introns.
cession of nucleotides linked through phosphodiester bonds
In addition, there are flanking sequences in the 59 and 39 ends
between the 59 position of the deoxyribose of one nucleotide
of the coding sequences that often contain important regula-
and the 39 position of the sugar moiety of the adjacent nucleo-
tory elements that control the expression of the gene.
Genes are arrayed in a linear fashion along chromosomes,
Conflict-of-interest disclosure: Dr. Graubert declares no com which are long DNA structures complexed with protein.
peting financial interest. Dr. Cook declares no competing financial Within chromosomes, DNA is bound in chromatin, a complex
interest. Dr. Schuettpelz declares no competing financial interest.
of DNA with histone and nonhistone proteins that “shield” the
Off-label drug use: Dr. Graubert: not applicable. Dr. Cook: not
applicable. Dr. Schuettpelz: not applicable. DNA from the proteins that activate gene expression.
| 1
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2 | Molecular basis of hematology
Figure 1-1 Flow of genetic information from DNA
to RNA protein. DNA is shown as a double-
stranded array of alternating exons (red) and
introns (pink). Transcription, posttranscriptional
processing by splicing, polyadenylation, and
capping are described in the text. The mature
transcript passes from the nucleus to the cytoplasm,
where it is translated and further modified to form
a mature protein. Reproduced with permission
from Hoffman R, Benz E, Silberstein L, Heslop H,
Weitz J, Anastasi J, eds. Hematology: Basic Principles
and Practice. 6th ed. Philadelphia, PA: Saunders
Elsevier, Inc.; 2013: 5.
Flow of genetic information complex of proteins (50-100) and five small nuclear ribonu-
clear proteins (snRNPs). mRNA splicing is an important
Transcription
mechanism for generating diversity of the proteins produced
RNAs are mostly single-stranded molecules that differ from by a single gene. Some genes exhibit alternative splicing, a
DNA in two ways: by a sugar backbone composed of ribose process by which certain exons are included in or excluded
rather than deoxyribose, and by containing the pyrimidine from the mature mRNA, depending on which splice sequences
uracil rather than thymine. The first step in the expression of are used in the excision process. For example, this is the
protein from a gene is the synthesis of a premessenger RNA means by which some erythroid-specific proteins of heme
(pre-mRNA). The transcription of pre-mRNA is directed by synthesis (aminolevulinic acid [ALA] synthase) and energy
RNA polymerase II, which in conjunction with other pro- metabolism (pyruvate kinase) are generated, contrasting with
teins generates an RNA copy of the DNA sense strand. This the alternatively processed genes in the liver and other tissues.
transcribed mRNA is complementary to the DNA antisense This permits functional diversity of the products of the same
strand. The pre-mRNA contains the sequences of all of the gene and is one of several determinants of tissue specificity of
gene9s exons and introns. The introns are then removed by a cellular proteins. Mutations in the sequences of either introns
complex process called mRNA splicing. This process involves or exons can derange the splicing process by either creating or
the recognition of specific sequences on either side of the destroying a splice site so that the intron sequence is not
intron that allow its excision in a precise manner that main- removed or the exon sequence eliminated. Recently, recurrent
tains the exon sequence. The mRNA may then undergo mod- mutations in mRNA splicing factors have been identified in
ifications at the 59 and 39 ends (capping and polyadenylation, patients with myeloid malignancies. The effect of these muta-
respectively). Although RNA splicing was thought to be tions on splicing has not yet been elucidated. If abnormal
restricted to the nucleus, it also can occur in the cytoplasm of splicing results in a premature stop codon (nonsense muta-
platelets and neutrophils activated by external stimuli. tion), then a surveillance pathway known as nonsense-
Splicing of mRNA is a critical step in gene expression with mediated decay may result in degradation of the abnormal
important implications for understanding hematologic mRNA. This mechanism generally applies to stop codon
disease. Splicing is controlled by the spliceosome, a large mutations in the first one-third to one-half of the mRNA and
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Basic concepts | 3
works to prevent synthesis of mutant peptides. When muta- of somatic cells), each nucleated cell in an individual has the
tions occur in the last one-third of the mRNA molecule, same diploid DNA content. Consequently, biologic processes
abnormal peptides may be produced. are critically dependent on gene regulation, the control of
gene expression such that proteins are produced only at the
appropriate time within the appropriate cells. Gene regula-
Translation
tion is the result of a complex interplay of specific sequences
The mature mRNA is transported from the nucleus to the within a gene locus, chromatin, and regulatory proteins
cytoplasm, where it undergoes translation into protein. The (transcription factors) that interact with those sequences to
mRNA is “read” in a linear fashion by ribosomes, which are increase or decrease the transcription from that gene.
structures composed of ribonucleoprotein that move along DNA sequences that lie in proximity to and regulate the
the mRNA and insert the appropriate amino acids, carried expression of genes, which encode protein, are termed cis-
by transfer RNAs (tRNAs), into the nascent protein. The acting regulatory elements. Nearly all genes have a site for
amino acids are encoded by three base triplets called codons, binding RNA polymerase II that is within the first 50 bases
the genetic code. The four bases can encode 64 possible 59 to the structural gene and is called the promoter region.
codons; because there are only 20 amino acids used in pro- Other sequences that regulate the level of transcription of
tein sequences, more than one codon may encode the same the gene are located at less predictable distances from the
amino acid. For this reason, the genetic code has been termed structural gene. Such sequences may increase (enhancers)
degenerate. An amino acid may be encoded by more than or decrease (silencers) expression. A special type of enhancer
one codon; however, any single codon encodes only one is locus control region (LCR), which was first and best
amino acid. The beginning of the coding sequence in mRNA defined in the β-globin cluster of genes on chromosome 11.
is encoded by AUG codon that has variable translation initia- It is located approximately 50 kilobases (kb) upstream from
tion activity determined by the neighboring nucleotide the β-globin gene, controls all genes in the β-globin locus,
sequences (Kozak sequence). In addition, there are three ter- and also has a strong tissue-specific activity (erythroid-
mination codons (UAA, UAG, and UGA) that signal the end specific).
of the protein sequence. Control of gene expression is exerted through the interac-
Single base-pair alterations in the coding sequence of tion of the cis-acting elements described previously with
genes may have a range of effects on the resultant protein. proteins that bind to those sequences. These nuclear DNA
Because the genetic code is degenerate, some single base-pair binding proteins are termed trans-acting factors or tran-
changes may not alter the amino acid sequence, or they may scription factors. Most of these proteins have a DNA bind-
change the amino acid sequence in a manner that has no ing domain that can bind directly to regulatory sequences
effect on the overall function of the protein; these are pre- within the gene locus; many of them contain common
dicted to be phenotypically silent mutations. Sickle cell dis- motifs, such as zinc-fingers or leucine zippers, which are
ease, however, is an example of a single base-pair change shared by many transcription factors. In addition, they fre-
(point mutation) resulting in an amino acid alteration that quently have unique domains that allow them to interact
critically changes the chemical characteristics of the globin with other transcription factors. Thus, a complex pattern is
molecule. Other mutations may change a codon to a termi- emerging whereby the expression of different transcription
nation codon, resulting in premature termination of the pro- factors, which may interact both with one another and with
tein (nonsense mutation). Finally, single or multiple base-pair specific regions of DNA to increase or decrease transcrip-
insertions or deletions can disrupt the reading frame of tion, determines the unique tissue, and stage-specific expres-
genes. These frameshift mutations render the gene incapa- sion of the genes within a given cell.
ble of encoding normal protein. These latter two abnormali-
ties account for some β-thalassemias and for polycythemia
Epigenetics
due to a gain of function in the erythropoietin receptor. Clin-
ically important mutations also may occur in the noncoding For a gene to be expressed, chromatin must be unwound and
region of genes, such as in the regulatory elements upstream the DNA made more accessible to regulatory proteins. This is
of the initiation codon or within intronic splicing sites. controlled by epigenetic processes, or modifications to the
genome that regulate gene expression without altering the
underlying nucleotide sequence. These changes may be
Control of gene expression
modulated by nutrition or drugs and may be heritable. Epi-
With the exception of lymphocytes (which undergo unique genetic modulation of gene expression was first recognized
changes in the DNA encoding immunoglobulin or the T-cell in studies of glucose-6-phosphate dehydrogenase (G6PD), a
receptor) and germ cells (which contain only half of the DNA protein encoded by an X-linked gene. Ernest Beutler deduced
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4 | Molecular basis of hematology
the principle of random embryonic X chromosome inactiva- region contains multiple genes associated with Prader-Willi
tion from studies of G6PD deficiency. His observations and syndrome, which are imprinted on the maternal allele. Thus,
the studies of Mary Lyon and Susumu Ohno on the mecha- maternal inheritance of a mutation or deletion in UBE3A
nism of dosage compensation in mammals led to an under- removes the single active copy of the gene and results in
standing of X chromosome inactivation in females. This was Angelman syndrome, and paternal inheritance of deletions
the first example of stochastic epigenetic silencing in humans, in this region remove the only active copies of the Prader-
demonstrating that human females are mosaics of the activ- Willi–associated genes and result in Prader-Willi syndrome.
ity of X chromosome–encoded genes. Using this principle in As DNA methylation modulates gene activity, aberrant
tumor tissue derived from females led to early demonstra- methylation may contribute to cancer. For example, in one
tions that neoplastic diseases are, for the most part, clonal. form of hereditary colorectal cancer, methylation of the pro-
Two common forms of epigenetic changes are DNA methyl- moter region of the MLH1 gene, whose protein product
ation and histone modifications. repairs damaged DNA, results in colon cancer. Likewise,
methylation-associated silencing of the DNA repair gene
BRCA1 is associated with breast and ovarian cancers, and
DNA methylation
hypermethylation of the promoter of the DNA repair gene
In addition to being complexed with protein, the DNA of MGMT correlates with improved clinical outcomes in patients
inactive genes is modified by the addition of methyl groups with gliomas treated with Temozolamide. Small molecule
to cytosine residues. Methylation normally occurs through- inhibitors of DNA methyltransferases (eg, 5-azacitidine,
out the genome. It is generally a marker of an inactive gene, decitabine) are used in the treatment of hematologic disorders
and changes in gene expression often can be correlated with that are characterized by aberrant DNA methylation (eg, myelo-
characteristic changes in the degree of methylation of the 59 dysplastic syndrome [MDS], acute myeloid leukemia [AML]).
regulatory sequences of the gene. This type of epigenetic
modification is performed by enzymes called DNA methyl-
Histone modification
transferases and is associated with alterations in gene expres-
sion and processes, such as X chromosome inactivation, Histones are DNA packaging proteins that organize DNA
imprinting, and carcinogenesis. into structural units called nucleosomes. Octamers of the
Monozygotic twins accumulate different methylation pat- core histones—H2A, H2B, H3, and H4—make up the nucleo-
terns in the DNA sequences of their somatic cells as they age, some around which 147 bp of DNA is wrapped, and histone
increasing phenotypic differences. Lifestyle disparities, espe- H1 binds the “linker” DNA between nucleosomes. Histones
cially smoking, result in even greater differences in their are subject to multiple modifications, including methylation,
DNA methylation patterns. Thus, despite having identical acetylation, ubiquitination, phosphorylation, and others. The
DNA sequences, twins become increasingly dissimilar particular combination of histone modifications at any given
because of epigenetic changes that result in different expres- locus is thought to confer a “histone code,” regulating pro-
sion of their identically inherited genes. cesses such as gene expression, chromosome condensation,
Mendelian genetics is based on the principle that the phe- and DNA repair. Like methylation, histone modifications
notype is the same whether an allele is inherited from the regulate gene activity and therefore disruptions of the normal
mother or the father, but this does not always hold true. Some pattern of these modifications can contribute to cancer and
human genes are transcriptionally active on only one copy of other diseases. For example, hypoacetylation of histones H3
a chromosome (such as the copy inherited from the father), and H4 are associated with silencing of the cell cycle regulator
whereas the other copy of the chromosome inherited from p21WAF1, a gene whose expression is reduced in multiple
the mother is transcriptionally inactive. This mechanism of tumor types. Small molecule inhibitors of the enzyme that
gene silencing is known as imprinting, and these transcrip- removes acetyl groups from histone tails (histone deacety-
tionally silenced genes are said to be “imprinted.” When genes lases) are being tested in a variety of hematologic malignan-
are imprinted, they are usually heavily methylated in contrast cies, and the histone deacetylase inhibitor Vorinostat is used
to the nonimprinted copy of the allele, which typically is not in the treatment of cutaneous T-cell lymphoma.
methylated. A classic example of imprinting is the inheri-
tance of Prader-Willi and Angelman syndromes, which are
Noncoding RNAs
associated with a 4-megabase (Mb) deletion of chromosome
15. This region contains the gene associated with Angelman It has been estimated that only approximately 1%-2% of the
syndrome, UBE3A, encoding a ligase essential for ubiquitin- genome encodes protein, but a much larger fraction is tran-
mediated protein degradation during brain development. scribed. This transcribed RNA that does not encode protein
This gene is imprinted on the paternal allele. In addition, the is referred to as noncoding RNA (ncRNA) and is grouped
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Basic concepts | 5
into an increasingly large number of different classes, includ- expression of miR-21, for example, is associated with poor
ing microRNAs, small nucleolar RNAs (snoRNAs), small prognosis in chronic lymphocytic leukemia, and higher
interfering RNAs (siRNAs), Piwi-interacting RNAs (piR- expression of miR-29b is associated with clinical response to
NAs), and many others. Although each of these classes of decitabine in older adults with AML.
ncRNAs differs in size, biogenesis pathway, and specific func-
tion, they share a common ability to recognize target nucleo-
Molecular basis of neoplasia
tide sequences through complementarity and regulate gene
expression. The most well-described class of ncRNAs are Normal cellular growth and differentiation depends on the
microRNAs (miRs), whose biogenesis pathway is illustrated precise control of gene expression, and alterations in the quan-
in Figure 1-2. Following transcription, a portion of this RNA tity or timing of gene expression can affect the survival and
(the pri-microRNA) forms hairpin loops that are cleaved by function of a cell. When such alterations occur in certain types
the enzymes Drosha and Dicer into short 21- to 23-bp of genes known as oncogenes or tumor suppressor genes, the
double-stranded RNAs. These short double-stranded RNAs cell may gain abnormal growth or survival properties, and
contain both sense strands and antisense strands that corre- accumulations of such mutations may lead to cancer.
spond to coding sequences in mRNAs. These mature miRs
then are incorporated into a larger complex known as a RISC
Oncogenes
(RNA-induced silencing complex). The miR is then unwound
in a strand-specific manner, and the single-stranded RNA Oncogenes are genes that have the potential to cause cancer,
locates mRNA targets by Watson–Crick base pairing. Gene and they arise from mutations in their normal counterparts
silencing results from cleavage of the target mRNA (if there termed proto-oncogenes. Proto-oncogenes generally code
is complementarity at the scissile site) or translational inhi- for proteins or ncRNAs that regulate such processes as prolif-
bition (if there is a mismatch at the scissile site). This gene- eration and differentiation, and activating mutations or
silencing pathway is known as RNA interference. Recent epigenetic modifications that increase the expression or
studies suggest that as much as 20% of cellular RNA is regu- enhance the function of these genes confer a growth or sur-
lated by RNA interference. As mediators of gene expression, vival advantage to a cell. The first described oncogene,
miRs and other ncRNAs play regulatory roles in development termed SRC, was discovered in the 1970s and is a member of
and differentiation, and they also are expressed in a tissue- a family of tyrosine kinases that regulate cell proliferation,
specific manner. Dysregulation or mutations in ncRNAs are motility, adhesion, survival, and differentiation. Activating
associated with various diseases, including cancer. Increased mutations in the SRC family kinases are associated with the
Figure 1-2 MicroRNA production. Production
of microRNA begins with transcription of the
microRNA gene to produce a stem-loop structure
called a pri-microRNA. This molecule is
processed by Drosha (cropping) to produce the
shorter pre-microRNA. The pre-microRNA is
exported from the nucleus; the cytoplasmic Dicer
enzyme cleaves the pre-microRNA (dicing) to
produce a double-stranded mature microRNA.
The mature microRNA is transferred to RISC
(RNA-induced silencing complex), where it is
unwound by a helicase. Complementary base
pairing between the microRNA and its target
mRNA directs RISC to destroy the mRNA (if
completely complementary) or halt translation (if
a mismatch exists at the scissile site). Reproduced
with permission from Murphy, et al, eds.
Molecular Biology of the Cell. 5th ed. New York,
NY: Garland Science/Taylor & Francis LLC; 2008.
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6 | Molecular basis of hematology
pathogenesis of multiple types of neoplasias, including can- to 39 on opposite strands). The DNA is cleaved by the enzyme
cers of the colon, breast, blood, head and neck, and others. on both strands at the site of the recognition sequence. After
Another classic example of an oncogene is the BCR-ABL1 restriction endonuclease digestion, DNA fragments may be
fusion gene found in chronic myelogenous leukemia (CML). separated by size using agarose gel electrophoresis, with the
This fusion results from a translocation between the BCR smallest fragments running faster (closer to the bottom of
gene on chromosome 9 and the ABL1 proto-oncogene on the gel) and the largest fragments moving more slowly
chromosome 22, and confers constitutive activation of ABL1 (closer to where the samples were loaded). DNA can be visu-
and enhanced cell proliferation. Pharmacologic targeting of alized in the gel by staining with ethidium bromide, a chemi-
the activity of oncogenes, such as the use of the tyrosine cal that inserts itself between the DNA strands and fluoresces
kinase inhibitor Imatinib to treat CML, can be an effective upon exposure to ultraviolet light. A desired fragment of
therapeutic approach. DNA may be isolated and then purified from the gel. Some
restriction enzymes generate overhanging single-stranded
tails, known as “sticky ends.” Complementary overhanging
Tumor suppressors
segments may be used to join, or ligate, pieces of DNA to one
In contrast to oncogenes, tumor suppressors are genes that another (Figure 1-3). These methods form the foundation of
encode for proteins or ncRNAs whose normal function is to recombinant DNA technology.
inhibit tumor development through the promotion of such
processes as apoptosis, DNA repair, cell cycle inhibition, cell
Polymerase chain reaction
adhesion, and others. Loss of the expression or function of
these genes is associated with cancer, and generally both cop- The polymerase chain reaction (PCR) is a powerful tech-
ies of the tumor suppressor gene must be altered to promote nique for amplifying small quantities of DNA of known
neoplasia. Thus, most tumor suppressors follow the “two-hit sequence. Two oligonucleotide primers are required; one is
hypothesis” proposed by Alfred Knudson in his study of the complementary to a sequence on the 59 strand of the DNA to
retinoblastoma-associated tumor suppressor gene RB1. This be amplified and the other is complementary to the 39 strand.
gene encodes a protein that functions to regulate cell cycling The DNA template is denatured at high temperature;
and survival. Because both copies of the gene must be the temperature then is lowered for the primers to be
mutated for retinoblastoma to manifest, individuals that annealed to the DNA. The DNA then is extended with a
inherit a mutant allele (requiring just one more “hit” in the temperature-stable DNA polymerase (such as Taq poly-
remaining normal allele for loss of gene function) generally merase), resulting in two identical copies of the original
develop disease earlier than those that must acquire “hits” in DNA from each piece of template DNA. The products are
both alleles. Familial cancer syndromes often result from the denatured, and the process is repeated. The primary product
inheritance of heterozygous mutations in tumor suppressor of this reaction is the fragment of DNA bounded by the two
genes. For example, Li-Fraumeni syndrome results from primers. Thus, small quantities of input DNA may be used to
inherited mutations in the cell cycle regulator TP53 and is synthesize large quantities of a specific DNA sequence. This
associated with the early onset of multiple tumor types, technique has superseded many blotting techniques for pre-
including osteosarcoma, breast cancer, leukemia, and others. natal diagnosis and cancer diagnostics. Using multiple
When mutations occur in the remaining normal allele, primer pairs in the same reaction, multiplex PCR can effi-
termed “loss of heterozygosity,” tumor growth is initiated. ciently amplify several fragments simultaneously.
More recently, loss of just one copy of a gene (“haploinsuffi- Reverse transcriptase PCR (RT-PCR) is a modification of
ciency”) has been shown to contribute to cancer develop- the PCR technique that allows for the detection and amplifi-
ment (eg, RPS14 haploinsufficiency in MDS). cation of expressed RNA transcripts. Complementary DNA
(cDNA) is generated from RNA using reverse transcriptase,
an enzyme that mediates the conversion of RNA to DNA.
Analytic techniques The resultant cDNA is then subjected to routine PCR ampli-
fication. Because cDNA is generated from processed mRNA
Digestion, amplification, and separation of
transcripts, no intronic sequences are obtained. RNA is
nucleic acids
much less stable than DNA; thus, amplification of mRNA
DNA may be cut, or digested, into predictable, small frag- from tissue or blood requires careful preservation of source
ments using restriction endonucleases. Each of these bacte- tissue or blood samples.
rially derived enzymes recognizes a specific sequence of 4-8 Quantitative PCR is another modification of the PCR
bp in double-stranded DNA. These recognition sequences technique. The most commonly used method is real-time
are usually palindromic (ie, they read the same sequence 59 PCR, in which a fluorogenic tag is incorporated into an
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Analytic techniques | 7
(a) Restriction enzyme digestion
Enzyme Recognition sequence Digestion products Overhang
EcoRI 5' GAATTC 3' 5' G3' 5'AATTC 3'
5'
3' CTTAAG 5' 3' CTTAA5' 3'G 5'
SacI 5' GAGCTC 3' 5' GAGCT3' 5'C 3'
3'
3' CTCGAG 5' 3' C5' 3'TCGAG 5'
PvuII 5' CAGCTG 3' 5' CAG3' 5'CTG 3' Blunt
3' GTCGAC 5' 3' GTC5' 3'GAC 5'
(b) Ligation of “sticky ends”
Figure 1-3 Restriction endonuclease GAATTC GAATTC
digestion. (a) Diagram of typical restriction
CTTAAG CTTAAG
enzyme recognition sequences and the
pattern of cleavage seen upon digestion with Cut with EcoRI
that enzyme. (b) Means by which restriction
enzyme can be exploited to form G AATTC G AATTC
recombinant proteins. Digestion of the two CTTAA G CTTAA G
fragments with the enzyme EcoRI results in
four fragments. Ligation with DNA ligase DNA ligase
can regenerate the original fragments, but it
GAATTC GAATTC
also can result in recombinant fragments in
which the 59 end of one fragment is ligated CTTAAG CTTAAG
to the 39 end of the second fragment. This
GAATTC GAATTC
recombinant DNA then can be used as a
template for generation of recombinant CTTAAG CTTAAG
protein in expression vectors.
oligonucleotide that will anneal to the internal sequence of contaminating DNA or RNA from other sources can cause
the Taq DNA polymerase-generated PCR product. This tag false-positive results. Clinical laboratories that use PCR for
consists of a fluorescent “reporter” and a “silencing” critical diagnostic tests require elaborate quality assurance
quencher dye at opposite ends of the oligonucleotide. When protocols to prevent inappropriate diagnosis. Equally trou-
annealed to the internal sequence of the PCR product, fluo- blesome can be false-negative results that result from inap-
rescence from the reporter is quenched because the silencer propriate primer design, degraded RNA, or inappropriate
is in proximity. After completion of each cycle of PCR temperature parameters for the annealing of primers.
amplification, the reporter is not incorporated in the prod- The amplified sequence of interest then can be rapidly
uct, but is cleaved by Taq DNA polymerase (because this evaluated for presence of mutation(s) by direct sequencing,
enzyme also has exonuclease activity). This fluorogen tag is restriction enzyme digestion (if a suitable enzyme that dis-
released, generating a fluorescent signal (Figure 1-4). Real- criminates between mutant and wild-type alleles is avail-
time PCR detects the number of cycles when amplification able), allele-specific PCR (discussed later in this chapter), or
of product is exponential and expresses this as a ratio to other techniques.
standard housekeeping RNA, such as ribosomal RNA or
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
Hybridization techniques
mRNA. This number can be converted to the number of
molecules of mRNA present in the test sample. This tech- DNA is chemically stable in the double-stranded form. This
nique is used widely to measure minimal residual disease or tendency of nucleic acids to assume a double-stranded struc-
to monitor clearance of BCR/ABL transcripts in patients ture is the basis for the technique of nucleic acid hybridiza-
treated with tyrosine kinase inhibitors. tion. If DNA is heated or chemically denatured, the hydrogen
The power of PCR lies in its great sensitivity, but this is bonds are disrupted, and the two strands separate. If the
also a potential weakness because small amounts of denatured DNA is then placed at a lower temperature in the
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8 | Molecular basis of hematology
Figure 1-4 Real-time PCR. (a) Sample DNA (or
A cDNA) is denatured, and target-specific primers
are annealed to begin the PCR amplification
(shown for one strand). An oligonucleotide probe
complementary to a sequence within the PCR
product is included in the reaction. The probe
contains a fluorophore (‘R’) covalently attached to
the 59 end and a quencher (‘Q’) at the 39 end. As the
Taq polymerase extends the nascent strand, its 59 to
39 exonuclease activity degrades the probe, releasing
the fluorophore from the quencher and allowing
the fluorophore to fluoresce. An example of this
fluorescent readout is shown in panel (b), which
depicts the relative fluorescence intensity from
amplification of the BCR-ABL fusion transcript
(yellow line) to an endogenous control transcript
(green line) in a patient with chronic myelogenous
leukemia before and after treatment with a tyrosine
kinase inhibitor. The cycle number at which
fluorescence crosses a threshold (horizontal dotted
line) is inversely proportional to the amount of
template DNA or cDNA. Although the control
template is consistently detected throughout
therapy, the BCR-ABL transcript abundance is
lower at 1 month (higher cycle threshold) and
undetectable at 3 months into therapy.
absence of denaturing chemicals, the single-stranded species denatured, labeled probe dissolved in hybridization solution
will reanneal in such a way that the complementary sequences is incubated with the denatured Southern blot membrane,
are again matched and the hydrogen bonds reform. If the which contains single-stranded DNA corresponding to the
denatured DNA is incubated with radioisotope- or fluorogen- entire cellular DNA. By molecular hybridization, the probe
labeled, single-stranded complementary DNA or RNA, the will anneal to complementary sequences within the DNA
radiolabeled species will anneal to the denatured, unlabeled fixed to the membrane. After the membrane is washed to
strands. This hybridization process can be used to determine remove the excess unbound probe and the probe that has
the presence and abundance of an identical DNA species. hybridized nonspecifically to areas of low-sequence homol-
The technique of molecular hybridization is the basis for ogy, the membrane is exposed to radiographic film or a fluo-
Southern blotting and many other molecular techniques. rescence detection system. The resultant image will allow
To perform Southern blot analysis, DNA is isolated from visualization of the DNA fragment or fragments that repre-
peripheral blood, bone marrow, or tumor tissue. The total sent the gene of interest with sequence complementary to
cellular DNA is then digested with specific restriction the probe (Figure 1-5). Southern blotting may be used to
enzymes. This results in a wide range of fragments that may determine whether a gene is present or absent or whether it
be separated by size using agarose gel electrophoresis. Because has been grossly rearranged by deletion, insertion, or
the DNA will be digested into thousands of fragments, recombination.
genomic DNA will appear in the gel as a continuous smear. Restriction fragment-length polymorphism (RFLP)
The DNA in the gel is denatured by exposure to alkaline buf- analysis is a Southern blot–based technique with many use-
fer, and the resulting single-stranded species are transferred ful applications in hematology. Using this technique, inher-
and fixed to a nitrocellulose or nylon membrane. ited disease-associated alleles may be identified and traced by
Detection of a specific gene fragment requires the use of a the presence of inherited mutations or variations in a DNA
probe. A probe is a labeled, single-stranded fragment of sequence that create or abolish restriction sites. Rarely, a
DNA that is specific to the gene of interest. Probes can be single-base, disease-causing DNA mutation will coinciden-
produced from any portion of gene whose sequence is tally fall within a recognition sequence for a restriction endo-
known or that previously has been isolated, such as globin or nuclease. If a probe for the mutated fragment of DNA is
the immunoglobulin heavy- and light-chain genes. The hybridized to total cellular DNA digested with that enzyme,
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