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Molecular Biology Techniques Kristen J. Skogerboe Laboratory of Pathology, 1229 Madison Street, Seattle, Washington 98104 The application of DNA technology to the clinical laboratory is an area that has generated enthusiasm as well as speculation. Research discoveries in molecular biology has led to a greater understanding of genetic diseases, cancer, and infection. The 1991 application review in clinical chemistry appearing in this journal did not include a section on DNA analysis. Even now, the application of these technologies in the clinical laboratory is in its infancy. Issues such as technology, quality assurance, cost, and privacy have implications in the application of DNA testing. The intent of this section of the review is to highlight several important areas of DNA testing that have appeared in the literature since 1989 (up to October 1992). This section will broadly cover areas of clinical analysis that involve the analysis of DNA. Included in this categorization are techniques that may include DNA probe hybridization, electrophoresis, or a variety of in vitro amplification techniques designed to enhanceDNA detectability. The cited references are targeted toward those that have demonstrated clinical diagnosis or have focused on diagnostic clinical lahoratoryaspects of DNA testing. Given these constraints, the section is not intended to be comprehensive. The first portion of the review will highlight work demonstrating the use of developing technologies for DNA testing. Although some of the cited work may not show immediate clinical applications, many of the technologieswillclearly beimportantin therapidlydeveloping field. The second portion will concentrate on the applications of DNA probe technology to (1)genetic disease, (2) cancer, and (3) infectious disease diagnosis.
DEVELOPING ANALYTICAL APPROACHES TO CLINICAL DNA TESTING Because of new developments in molecular genetics the role of the clinical laboratory faces many potential changes. The predicted impact of molecular genetic technological advances on the clinical laboratory has been reviewed ( K l ) . New DNA analysis techniques are the subject of two reviews that address the need for new strategies and methods for detection of genetic diseases (K2, K3). One author has reviewed the current status of DNA hybridization assays and the relevance to laboratory medicine (K4,KS). The application of biotin-avidin probe labeling to biotechnolo has been described (K6). Quantification of nucleic acid g u n d to membranes using time-resolved fluorometry has been reported, with a demonstrated detection limit of 10 pg of target DNA (K7). A dot blot format using time-resolved detection with enzyme-amplified lanthanide luminescence has demonstrated a detection limit of 4 pg of nucleic acid target (K.8). Of great importance in the development of molecular techni ues and the application in the clinical laboratory are metho& for in vitro amplification of DNA. One technique, the polymerase chain reaction (PCR) has generated substantial enthusiasm for clinical diagnosis. ThetheoryofPCR andclinicalapplications has been reviewed (K9). Other work describes applications of PCR to forensic medicine (KlO). PCR is one of several DNA probe amplification methods discussed in a review of new techniques ( K I I ) . Other amplification technologies relevant to clinical chemistry have beendeveloped includingthe ligasechain reaction (K12,K13), Q-6 replicase (K14, K 1 8 , and chimeric cycling (K16). One of the challenges of applying DNA amplification techniques in the clinical laboratory is the development of rapid and accurate means of detecting the products of an amplification reaction. A colorimeteric DNA enzyme immunoassay method has been developed as a general method for detecting PCR products ( K l 7 ) . This method has demonstrated good detectability of DNA from the hepatitis B virus. Alternative strategies for detecting PCR products have focused on the development of a triple-helical capture assay (K18). ThisapproachusesPCRamplification chemistry that enables the production of triple-helical complexes. These complexes enable rapid sequence-specific detection without requiring blocking reagents, labor-intensive washing steps, 410R
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K M e n J. S k w h received a B S in chemsby in 1982 from Colorado Stale
Universilv. In t986sheremivedhsrPhD
in analytical chemistry from Iowa State
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University. Foilowing two years of postdoctoral trainlng in clinical chemisby In the Department of Laboratwy Medicine at the 'University of Washinglon. she accepted a fellowship in clinical molecular genetics to pursue her interest in genetic disease dmgnosis. In 1991, she was awarded diplomat status by the American Board of Clinical Chemistry. She is presently an associate laboratory director at the Lab& ( oratory of Pathology in Seattle, WA. In addlion to her interest In the application of Unique spectroscopic and chromatographic memods to clinical analysis, she is interested in developing techniques for clinical DNA analysis.
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or time for color development. Another uni ue means of detecting PCR products is through electrochem%minescence (ECL) (K19). This method relies on a reagent that exhibits ECL, used as a primer label in the PCR reaction. Methods for detection of human papilloma virus and the ras oncogene have been developed usin this approach. Other nonradioisotopic detection methofs for PCR products, suitable for automated clinical laboratory applications, have been reported (K20). Detection of PCR products in a dot blot assay using image analysis has been demonstrated with the human leukocyte antigen gene (K21). A factor limiting the application of DNA technology to the clinical laboratory is the development of methods for rapid specimen processing. Rapid sample preparation for detection of DNA viruses in human serum by PCR has been reported (K22). The direct PCR amplification of washed blood cells without nucleic acid extraction has heen developed (K23). The addition offormamide to PCR reactions has heen shown to dramatically improve the specificity of the reaction, while increasing the efficiency of the amplification (K24). DetectionofsmalldifferencesinDNAsequence is becoming increasingly important for the identification of mutations in the diagnosis of genetic diseases or pathogenic determinations. Methods for the rapid detection of point mutations or small sequence alterations include an assay based on single-strand conformation polymorphism (SSCP) for rapid detection of mutations using denaturinggelelectrophresis(K25). A hybrid technique called dideoxy fingerprintin is a method that uses dideoxy sequencing chemistry and 8SCP electrophoresis methods in combination (K26). Another method for rapidly detectin known single-base changes that is of interest to clinical &agnosis is based on PCR amplification of specific alleles. This method is based on allele-specific PCR amplification, where the reaction chemistry is desi ed so that only DNAofagivensequencewillbeamplified. !%e presence or absence of PCR product is used to assess whether a mutation is present (K27). The technical complexity of methods for DNA sequencing is a primary factor in the application of this technology in the clinical laboratory. However, progress in the analytical technology for automated sequencing is occurring so that the use of sequencing for diagnosing genetic disease is on the horizon. An automated PCR-based sequencing method has heen demonstrated for clinical investigation of the HLAgene (K28). Less developed innovations in DNA sequencing technology include the use of capillary gel electrophoresis combined with fluorescence detection. Work using this technology has shown the capability to sequence 500 basesih with a single capillary (K29). When a capillary array is used in a similar format, the estimated rate of DNA sequencing is 50 OOO bases/h (K30). Other work extending this concept has employed laser-induced fluorescence detection and a sheath flow cuvette to improve detectability (K31). The sensitivity of fluorescence is not the only spectroscopic approach being exploited for DNA sequencin A method using resonance ionization spectroscopy has flmonstrated
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applicability, where stable isotopes have been used to label DNA and are detected using time of flight mam spectometry (K32, K33). Electro horetic techniques remain a cornerstone of DNA analysis. [everal new developments in electrophoresis technology have potential a plication in the clinical laboratory. One important technidap lication is hi hlighted in a review of pulsed field gel electro goresis, a tecfmique used to ain better resolution of lar e D%A fragments (K34). Using p&ed field techniques, anotter group has develoDed a method for improvedseparation of nudeic acids that us& analyte velocity modulation in caDillarv electroDhoresis(K35).This method uses changes in-field" strength and direction to improve resolution of smaller DNA fragments. Capillary gel electrophoresis has been used to evaluate restriction fragments (K36). Affinity capillary gel electrophoresishas also been applied to DNA f r a p e n t detection (K37).In this method, an affinity ligand is mcorporated into a olyacrylamide el capillary to increase the selectivity of t e separation. bield strength gradients have also been a plied to enhance the separation of DNA by capillary gef electrophoresis for restriction fragments (K38)and PCR products (K39).Detection of nucleic acids in agarose gels has been the focus of work usin noninvasive imaging to quantify nanogram amounts of DN (K40).This paper reports the use of the electricbirefringence as the basis for im ing, negating the use of fluorescent dyes such as ethidium%omide, which can damage DNA for subsequent uses.
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DNA PROBES FOR GENETIC DISEASE DIAGNOSIS Dramatic discoveries in human genetics have given rise to the capability to provide DNA-based diagnostic testing for numerous genetic diseases. Some of these genetic diseases are rare, so clinical testing will remain in the realm of research or specialty laboratories. However, several of the discoveries have included genes that occur with a high enough frequency to justify larger scale clinicallaboratory testing. DNA testing in cystic fibrosis (CF) is a primary challenge for integrating genetic disease testing into a clinical laboratory (K41).With a frequency of the cystic fibrosis carrier state of 1in 25 in the Caucasian population, DNA carrier testing has the potential to be a widely ado ted clinical test. Numerous methods for CF testing have teen reported, including a method with technology aimed at carrier screening (K42).This method is based on reverse dot blot hybridization and the polymerase chain reaction and detects the most fre uent mutations responsible for CF. Alternatively,a metho8based on a DNA enzyme immunoassay using a colorimetric label has been reported for CF mutations (K43).This PCR-based method uses an anti double-stranded DNA antibody to detect the presence or absence of DNA hybrids and is adaptable to automation. Another interesting method for CF mutation analysis is based on solid-phase minisequencing and can be performed in a microtiter plate (K44). For other genetic disorders where there are ongoing screening programs, DNA testing may provide benefits for identification of unusual phenotypes and genotypes, or in family studies to identify asymptomatic carriers. Po ulation screening for Gaucher's disease has been roposef, and a method to identify common mutations in &is disorder has been reported (K45). This method, based on PCR and a color complementation assay, detects the most common mutation in the population studied. A PCR-DNA test for the relatively common recessive disorder, a-1-antit deficiency,has been developed (K46).A second m e t h o y z based on site-mediated mutagenesis has been extended to samples of dried blood collected on filter paper (K47). DNA-based testing for individuals predisposed to atherosclerosis and coronary heart disease is certainly relevant to clinical chemistry. Due to inheritance, certain individuals are susceptible to these disorders and can be identified by DNA testing. Using a DNA method called the amplification a technique based on refracto mutation s stem (ARMS), PCR, in%iduals w i d variants of the apoliprotein E gene can be identified (K48).This method relies on the lack of PCR amplification if a particular mutation is present, with PCR products evaluated by gel electrophoresis. A similar
method has been published to detect mutations of the apoliprotein B-100 gene (K49). Fragile X mental retardation is one of the most common causes of inherited mental retardation, with a male population incidence of about 1in 10oO. In 1991 the gene for fragile X was discovered, a finding that will likely make DNA testin the standard method for diagnosing this disorder and identifying carriers. A method based on agarose gel electrophoresis followed by Southern blot analysis has been reported (K50).Other groups are concentratingon developing teC~OlOW more easily adaptable to large-scale carrier screening by developing PCR-based assays (K51). Diagnostic testin for porphyria is an important component of many clinical clemistry departmenu. Although most current testing is based on detectin abnormal porphyrins or porphyrin precursors, this type o f testing has limitations. Advances in the understanding of the molecular defects in orphyria indicate that DNA testin for orphyria may gecome an adjunct to the di osis o porp yria. Identification of mutations and thezvelopment of a DNA-based assay for mutations associated with acute intermittent This method is based on porphyria has been reported (K52). denaturin gradient gel electro horesis, an important molecular biaogy technique that igntifies small differences in DNA sequences. Similar technology has been applied in the identification of mutations associated with retinitis igmentosa (K53), a disorder that is characterized by night b indness and visual loss. Prenatal diagnostic testing for fetal abnormalities is a rowing area of clinical analysis. Historically, most testing gas not been performed in clinical chemistry laboratories. However, as im rovements in technology and automation occur, this may tegin to change. One exam le of a chemical measurement that could be performed in a cEnical laboratory is the use of PCR to molecularly diagnose numerical sex chromosome abnormalities (K54).Other research in this area has focused on the isolation of fetal cells from maternal blood and subsequent identification of numerical chromosome abnormalities without requiring an invasive procedure such as amniocentesis (K55). Identification of enetic disease in the newborn eriod is important in a num%er of disorders, prompting wilespread newborn screening programs. While most of the disorders can be diagnosed by protein-based testing, DNA testing may have advantages for either the diagnosis or the confirmation of some genetic diseases. One group has used PCR to identify molecular defects that cause congenital adrenal hyperplasia (K56).DNA probe methods for hemoglobin abnormalities have also been developed, including one method for the identification of newborns with hemoglobin E disease (K57). Another group has used a color complementation assay to diagnose complex hemoglobin genotypes (K58).Chemical cleavqe mismatch has been used to screen for &thalassemia mutations, culminating in the development of a method that could also be used for prenatal di osis (K59).A study comparing the results of DNA a n T s i s with hemoglobin electrophoresisin the analysis of newborn blood samples has also been published (K60).DNA testing for a-thalassemia has also been reported and is important for couples where both spouses are identified as carriers (K61). Many immunolo ic disorders have a genetic component, leading to the speculation that DNA testing may someday be widespread in the identification of individuals at risk for developin disease. Allelic forms of the human leukocyte anti en (ALA) DQ @-chaingene have been recognized as a marEer for insulin-dependent diabetes mellitus (IDDM) susceptibility. Trucco et al. have re orted a method for the ra id detection of alleles associatetf with IDDM based on PER and electro horesis (K62).Silver staining of amplified HLA DNA has $80 been developed, with detectability of at least 8 ng of DNA (K63).
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DNA DIAGNOSIS AND CANCER The connection between genetics and cancer is becoming better understood. Familial inheritance and susceptibility to a variety of different cancers is a tremendously active area of clinical scientific investigation. How DNA testing will ultimately fit into the diagnosis and treatment of cancer is yet to be determined. However, clinicians are already using ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993
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the DNA diagnostic laboratory to identify individuals with mutations in the familial adenomatous polyposis gene (K64). This work is based on PCR analysis of DNA sequences on chromosome 5 and has been used to study mutations in some colon cancer patients.' Another DNA test that has become an important clinical tool in the diagnosisof cancer is analysis of rearranged DNA sequences in patients with certain types and lymphomas (K66).Sensitiveanalytical of leukemia (K65) techniques based on PCR enable clinical cancer laboratories to detect DNA sequences associated with residual myeloma or leukemia in patients with clinical parameters indicating complete remission (K67).Multiplex PCR, a technique that simultaneously amplifies more than one segment of DNA, is the basis for a method developed to look at point mutations of ras protooncogenes, genes associated with tumorogenesis in a wide variety of human cancers (K68).
DNA ANALYSIS IN INFECTIOUS DISEASE DIAGNOSIS The strengths and pitfalls of molecular ap lications to infectious disease diagnosis have been reviewed ($69). Clearly there are limitations to the ap lication of this technology, but the future still seems very !right. The primary benefit of DNA analysis of bacterial or viral DNA is the proposition that the pathogen can be detected directly. If direct detection can be achieved,this negates the need to culture the pathogen or to detect antibodies to the pathogen. The selectivity afforded by detecting selected regions of pathogenic DNA sequences is also attractive. DNA hybridization has been combined with bioluminescence to form the basis for an automated instrumentation to detect bacteriuria in urine samples (K70).Signal amplification has been used to detect pathogens that are present in low copy number. Although PCR is the technique that has been most utilized, other methods have been under development. The in vitro amplification technique, Q-@replicase has been used to amplify viral nucleic acid, showing a 1-fg detection limit for RNA from the human immunodeficiency virus (HIV) (K71).Other work has concentrated on the detection of multiple patho ens simultaneously. Using Multiplex PCR, a variety of fifferent forms of the human papillomavirus (HPV) have been detected (K72). The detection and diagnosis of hepatitis is an important job of the clinical laboratory. With the varied physiologic response to hepatitis infection, and multiple forms of the virus, diagnosis and monitoring of hepatitis is complicated. Advances in DNA probe technology have aided the clinician with the care and treatment of atients with hepatitis. The presence of hepatitis B virus (IfBV) DNA in sera is thought to he the most definitive marker of an active viral infection. Several direct methods for detection of DNA from the hepatitis B virus have been develo ed. A nonisotopic method based on a sulfonated DNA proie detected by a sandwich immunoenzymatic reaction detects virus at a level of 2.5 ng/L (K73).Other work has compared the sensitivity and specificity of phos horus-32-, biotin-, alkaline phosphatase-, or sulfone-labele! DNA probes (K74).The conclusion of this work is that radiolabeling is still the most sensitiveand reliable detection of hepatitis B DNA. For enhanced detectability, PCR has been used to detect HBV in dried blood spots, detecting as little as 10 virus particles by this technology
(K75).
Direct detection of nucleic acids from the hepatitis C virus (HCV) may greatly facilitate early diagnosis of infection and may be advantageous in the monitorin of interferon therapy. An immunoassay for detection of PCkamplified HCV has been developed based on an antibod that selectively recognizesdouble, but not single-stranded6NA (K76).Another method has been developed to determine the HCV subtype and has been used in epidemiologic studies (K77).Other researchers have evaluated RNA extraction methods and PCR for the detection of enterically transmitted non-A, non-B hepatitis virus (HEV) for screening clinical specimens (K78). Diagnosis of HIV infection has historically been done by antibody testing. Direct detection of the virus without requiring seroconversion is an attractive alternative for detecting infected individuals. Three nonradioisotopic PCR methods for detecting HIV DNA have been compared, finding that the techniques are sensitive and specific for clinical 418R
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samples (K79). In other work, blot hybridization and microtiter plate methods for detecti HIV amplified by PCR have been compared, showing some%scordance between the two methods (K80).Interestingly, heparin has been shown to inhibit HIV virus ene amplification,indicating that certain anticoagulanta shoukd be used for collection of clinical samples where PCR is to be performed (K81).Susceptibility testing to measure the resistance of HIV subtypes to antiviral drugs has been performed using quantitative PCR DNA method (K82). Clinical methods for identifying mycobacterium have typically been time consuming,requiring lengthy culture steps for the identification of the organism. DNA probe technology has been shown to provide a means of detecting Mycobacterium tuberculosis in one working day using a sucrose centrifu ation step to purify the sample followed by PCR (K83).leestriction endonucleasedigestion followed by pulsed field gel electrophoresis has been used for subspecies identification of Mycobacterium avium complex (K84).A ve simple method based on PCR and reverse dot blot hybriz ization has been developed for mycobacteria and is applicable for implementation in the clinical laboratory (K85). Human papillomavirus (HPV) is a patho en that has been associated with cervicalcancer. Although cknical studies are still underway, it has been suggested that detection of individualswith HPV infection may be a beneficial com nent of screening programs for cervical cancer. A methoEased on Southern transfer hybridization and dot fiiter hybridization has been used to detect and classify the viral subtypes in HPV infection (K86).PCR-based methods for HPV have also been reported using sulfonated primers (K87). A variety of DNA-based methods for the detection of other viral or microbial pathogens have been developed, with great potential in the clinical laborato . One group has compared the use of biotinylated DNA anrRNA probes for the rapid detection of varicella-zoster virus (K88). Detection of speciesspecific DNA fragments has formed the basis for an assay to detect the presence of Candida albicans by PCR (K89).DNA testing has been shown to be useful in the molecular di of Lyme disease (K90).Electrophoretic evaluation products has been used to detect cytomegaloviral strains (K91).Finally, a direct chemiluminescence dot blot assay for Parovirus has been developed (K92). LITERATURE CITED (KI) WIIkmson, R. C//n.Chem. 1989, 35, 2165-2188. (K2) Coutelle, C. &md. & & e m . Acta 1991. 50, 3-10. (K3) Forrest, S.; Cotton, R. 0. H. Mol. Bkl. M d . 1990, 7, 451-459. (K4) Mamandls, E. P. C//n. Chem. Acta 1990, 104, 19-50. (K5) Memandis, E. P. Clln. Bbchem. 1990, 23, 437-443. (K6) MaI'IWndls, E. P.; ChrlStOpOUlOS, T. K. CUn. Chem. 1991, 37, 825-636. (K7) CMstopoulos,T. K.; Mamandls E. P.; Wilson 0. Nw&b A& Res. 1991. IO, 6015-6019. (K8) Templeton, E. F. Q.; Wong, H.E.; Evangellsta, R. A.; Qanger, T.; Pollak, A. Clln. Chem. 1991, 37, 1506-1512. (K9) Remlck, D. G.; Kunkel, S. L.; Holbrook, E. A.; Hanson, C. A. Am. J. Clln. PeW. 1990, 03, S49-S54. (K10) Reynolds, R.; Sensabaugh, Q.; Blake, E. Ana/. Chem. 1991, 63, 2-15. (K11) Blrkenmeyer, L. 0.;Mushahwar, I. K. J. V M . Meethods 1991, 35, 117126. (K12) Backman, K. CUn. Chem. 1992, 38. 457-458. (K13) Barany, F. Roc. Mtl. Aced. W. U S A . 1991, 88, 189-193. (K14) Cahill. P.; FOster, K.; Mahan, D. E. CUn. Chem. 1991, 37,1482-1485. (K15) Kramer. F. R.; Uzardl, P. M.; Tyaol, S. CUn. Chem. 1992, 38,456-457. (K16) Duck, P.; Alvarado-Urblna, G.; Burdlck, B.; Colller, B. B&TectIn@h9s 1990, 0, 142-148. (K17) Mantero, G.; Zonaro, A.; Albertlni, A.; Bertolo, P.; Prlml, D. Cln. Chem. isei. 37.422-429. (K18) Vary, C. P. H. CUn. Chem. 1992, 38, 667-694. (K19)Kenten,J.H.;Caaedei.J.;LInk,J.;Lupold,S.;WIlky,J.;Powell,M.;R~, A.; Massey, R. CNn. Chem. 1991, 37. 1626-1632. (K20) Wollnsky, S. M.; Drew, J. 8.;Mllman, Q.; Hoff, R.; Dragon. E. A.; nul, J.; Otto, P.; Oupta, P.; Farzadegan, H.; Whetsell, A. J. Clln. Chem. 1992, 38, 459-460. (K21) Ludwig, M.; Hartzman, R. J. Anal. Chem. 1992, 84, 2878-2681. (K22) Frlckhofen, N.; Young, N. S. J. V M . Methods 1891, 35, 65-72. (K23) Nwdvaag, B. Y.; Husby, Q.; Ei-Qewely, M. R. BloTechnIqw 1992, 12, 490-493. (K24) Sarkar. G.; Kapelner, S.; Sommer, S. S. Nudeic Aclds Res. 1990, 18, 7465. (K25) Lw, H. H.;LO, W. Ye;ChOO. K. B. Anal. Bkchem. 1992,205,289-293. (K26) Sarkar, Q.; Yoon. H. S.; Sommer, S. S. 5snomks 1992, 73,441-443. (K27) Sommer, S. S.; Qosrbach. A. R.; Bottema, C. D. K. BloTectIniques1992, 12, 82-87. (K28) MctkMe, L. J.; Koepf, S. M.; (Ybbs, R. A,; Saleet, W.; Mayrand. P. E.; Hunkaplller, M. W. Kronlck, M. N. C//n. Chem. 1989, 35, 2196-2201.
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