Page 42 - Hematology_ Basic Principles and Practice ( PDFDrive )

14 Part I Molecular and Cellular Basis of Hematology

Northern blotting represents an analogous blotting procedure polymerases is that they retain activity in a reaction mix that is
used to detect RNA. RNA cannot be digested with restriction repeatedly heated to the high temperature needed to denature the
enzymes (which cut only DNA); rather, the intact RNA molecules DNA strands into the single-stranded form. Microprocessor-driven
can be separated according to molecular size by electrophoresis DNA thermocycler machines can be programmed to increase tem-
through the gel (mRNAs are 0.5 to 12 kilobases in length), transferred peratures to 95°C to 100°C (203°F to 212°F) (denaturation), to cool
onto membranes, and probed with a DNA probe. In this fashion, the the mix to 50°C (101°F) rapidly (a temperature that favors oligonucle-
presence, absence, molecular size, and number of individual species otide annealing), and then to raise the temperature to 70°C to 75°C
of a particular mRNA species can be detected. (141.4°F to 151.5°F) (the temperature for optimal activity of the
Western blotting is a similar method that can be used to examine thermophilic DNA polymerases). In a reaction containing the test
protein expression. Cellular lysates (or another source of proteins) can specimen, the thermophilic polymerase, the primers, and the chemi-
be electrophoresed through a polyacrylamide gel so as to separate cal components (e.g., nucleotide subunits), the thermocycler can
proteins on the basis of their apparent molecular sizes. The resolved conduct many cycles of denaturation, annealing, and polymerization
proteins can then be electrically transferred to nitrocellulose mem- in a completely automated fashion. The gene of interest can thus be
branes and probed with specific antibodies directed against the amplified more than a millionfold in a matter of a few hours. The
protein of interest. As with RNA analysis, the relative expression DNA product is readily identified and isolated by routine agarose gel
levels and molecular sizes of proteins can be assessed with this method. electrophoresis. The DNA can then be analyzed by restriction endo-
nuclease, digestion, hybridization to specific probes, sequencing,
further amplification by cloning, and so forth.
Polymerase Chain Reaction
USE OF TRANSGENIC AND KNOCKOUT MICE TO DEFINE
The development of the polymerase chain reaction (PCR) was a
major breakthrough that has revolutionized the utility of a DNA- GENE FUNCTION
based strategy for diagnosis and treatment. It permits the detection,
synthesis, and isolation of specific genes and allows differentiation of Recombinant DNA technology has resulted in the identification of
alleles of a gene differing by as little as one base. It does not require many disease-related genes. To advance the understanding of the
sophisticated equipment or unusual technical skills. A clinical speci- disease related to a previously unknown gene, the function of the
men consisting of only minute amounts of tissue will suffice; in most protein encoded by that gene must be verified or identified, and the
circumstances, no special preparation of the tissue is necessary. PCR way changes in the gene’s expression influence the disease phenotype
thus makes recombinant DNA techniques accessible to clinical labo- must be characterized. Analysis of the role of these genes and their
ratories. This single advance has produced a quantum increase in the encoded proteins has been made possible by the development of
use of direct gene analysis for diagnosis of human diseases. Indeed, recombinant DNA technology that allows the production of mice
PCR analysis combined with direct DNA sequencing technologies that are genetically altered at the cloned locus. Mice can be produced
have largely supplanted restriction enzyme mapping and blotting that express an exogenous gene and thereby provide an in vivo model
strategies for many research and diagnostic applications. of its function. Linearized DNA is injected into a fertilized mouse
The PCR is based on the prerequisites for copying an existing oocyte pronucleus and reimplanted in a pseudopregnant mouse. The
DNA strand by DNA polymerase: an existing denatured strand of resultant transgenic mice can then be analyzed for the phenotype
DNA to be used as the template and a primer. Primers are short induced by the injected transgene. Placing the gene under the control
oligonucleotides, 12 to 100 bases in length, having a base sequence of a strong promoter that stimulates expression of the exogenous gene
complementary to the desired region of the existing DNA strand. in all tissues allows the assessment of the effect of widespread over-
The enzyme requires the primer to “know” where to begin copying. expression of the gene. Alternatively, placing the gene under the
If the base sequence of the DNA of the gene under study is known, control of a promoter that can function only in certain tissues (a
two synthetic oligonucleotides complementary to sequences flanking tissue-specific promoter) elucidates the function of that gene in a
the region of interest can be prepared. If these are the only oligonucle- particular tissue or cell type. A third approach is to study control
otides present in the reaction mixture, then the DNA polymerase can elements of the gene by testing their capacity to drive expression of
only copy daughter strands of DNA downstream from those oligo- a “marker” gene that can be detected by chemical, immunologic, or
nucleotides. Recall that DNA is double stranded, that the strands are functional means. For example, the promoter region of a gene of
held together by the rules of Watson–Crick base pairing, and that interest can be joined to the cDNA encoding green jellyfish protein
they are aligned in antiparallel fashion. This implies that the effect of and activity of the gene assessed in various tissues of the resultant
incorporation of both oligonucleotides into the reaction mix will be transgenic mouse by fluorescence microscopy. Use of such a reporter
to synthesize two daughter strands of DNA, one originating upstream gene demonstrates the normal distribution and timing of expression
of the gene and the other originating downstream. The net effect is of the gene from which the promoter elements are derived. Transgenic
synthesis of only the DNA between the two primers, thus doubling mice contain exogenous genes that insert randomly into the genome
only the DNA containing the region of interest. If the DNA is now of the recipient. Expression can thus depend as much on the location
heat denatured, allowing hybridization of the daughter strands to the of the insertion as it does on the properties of the injected DNA.
primers, and the polymerization is repeated, then the region of DNA In contrast, any defined genetic locus can be specifically altered
through the gene of interest is doubled again. Thus, two cycles of by targeted recombination between the locus and a plasmid carrying
denaturation, annealing, and elongation result in a selective quadru- an altered version of that gene (Fig. 1.9). If a plasmid contains that
pling of the gene of interest. The cycle can be repeated 30–50 times, altered gene with enough flanking DNA identical to that of the
resulting in a selective and geometric amplification of the sequence normal gene locus, homologous recombination can occur, and the
30
50
of interest to the order of 2 to 2 times. The result is a millionfold altered gene in the plasmid will replace the gene in the recipient cell.
or higher selective amplification of the gene of interest, yielding Using a mutation that inactivates the gene allows the production of
microgram quantities of that DNA sequence. a null mutation, in which the function of that gene is completely
PCR achieved practical utility when DNA polymerases from lost. To induce such a mutation, the plasmid is introduced into an
thermophilic bacteria were discovered; when synthetic oligonucle- embryonic stem cell, and the rare cells that undergo homologous
otides of any desired sequence could be produced efficiently, repro- recombination are selected. The “knockout” embryonic stem cell is
ducibly, and cheaply by automated instrumentation; and when DNA then introduced into the blastocyst of a developing embryo. The
thermocycling machines were developed. Thermophilic bacteria live resultant animals are chimeric; only a fraction of the cells in the
in hot springs and other exceedingly warm environments, and their animal contain the targeted gene. If the new gene is introduced into
DNA polymerases can tolerate 100°C (212°F) incubations without some of the germline cells of the chimeric mouse, then some of the
substantial loss of activity. The advantage of these thermostable offspring of that mouse will carry the mutation as a gene in all of