Molecular biology techniques

Thalassemia is one of the most common single-gene disorders. ..... Genet. 1994, 93, 16-20. (L23) Remes, A. M.; Hassinen, I. E.; Ikaheimo,M. J.; Herva,...
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Molecular Biology Techniques Kristen J. S k o g e h Laboratory of Pathology, 1229 Madison Street, SeatHe Washington 98104

This review covers an approximate two-year period from 1992 through 1994 and focuses on methods of DNA analysis for the purpose of clinical diagnosis. Since the 1992 review, there has been a rapid rate of discovery and understanding of genetic disease, leading to the Understanding that much of human disease has a foundation in genetics. Genes for a variety of genetic disorders have been cloned including, Huntington’s disease (LI), adult polycystic kidney disease (LZ), and Wilson’s disease (D), along with genes for hereditary forms of colon cancer (LA)and familial breast cancer 0.This short review is not intended to be comprehensive, but focuses on current or upcoming laboratory methods based on DNA analysis. The first portion of the review will cover applications of DNA probe technology to (1) genetic disease, (2) cancer, and (3) infectious disease diagnosis. The second part of the review will highlight developing technologies in molecular biology. Many of the papers cited in the second section describe pilot or demonstrationprojects that show potential to the area of clinical DNA analysis. Therefore, many of these applications are in the infancy and may, or may not, be adopted as useful and reliable methods. DNA ANALYSIS FOR GENETIC DISEASE DIAGNOSIS With the exception of newborn screening, widespread laboratory screening for most genetic diseases has not yet become commonplace,owing to ethical, legal, and technologicalconcerns. While individuals with a family history are being offered carrier testing for cystic fibrosis (CF), evaluation of mass screening methods is under review with an eye to the future (L6).Two methods for large-volume screening of cystic fibrosis heterozygotes have been compared (157). Technical suggestions to improve methods for CF testing include a gel matrix for improved resolution of polymerase chain reaction (PCR) products (Ls). Other improvements include demonstration of rapidcycle DNA amplification combined with allele specifk amplification to detect CF mutations (L9).A unique method of rapid screening has been demonstrated with the application of methodology based on mutation-specificartificial methylation sites. This is an isotopic method and involves sequence modification to create either mutant or wild-type methylation sites in the PCR product. After digestion with methylase and a tritiated DNA analogue, samples are counted for incorporation of tritium (L1O). Thalassemia is one of the most common single-gene disorders. Many states are adopting hemoglobin/thalassemia screening programs, but most of these are not DNA based. DNA methods for rapid detection of mutations will be valuable in providing better ways of diagnosing thalassemia. A new development is the demonstration of PCR-based methods for a-thalassemia (L11, LIZ). These papers describe unique multiplex primer sequences that enable the detection of large deletions of the a-globin gene. A direct genotyping method for ,&thalassemia based on PCR has been reported (LI3). Another group has developed a method for the detection of six mutations of the /3-globin gene, providing comprehensive scanning and rapid detection (LI4). The analysis of apolipoproteinshas become increasingly more important in the clinical laboratory. Apolipoprotein E has been

associated with familial Alzheimer’s disease (1515). PCR and isoelectric focusing methods for identification of apolipoprotein E polymorphisms were compared (LI6). Common and rare genotypes of the apolipoprotein E gene have been determined by PCR (L17). Other researchers have applied single-strand conformationpolymorphism (SSCP) analysisto the determination of apolipoprotein E genotypes. SSCP is a procedure used to screen for mutations in DNA fragments, where single-stranded PCR products have altered electrophoretic mobilites if conformational changes associated with mutations are present (L18).A robust strategy for screening and confirmation of defective apolipoprotein €3-100genes was reported, based on mutagen PCR, and restriction enzyme digestion (LI9). The amplification refractory mutation system (ARMS) is a method based on lack of PCR amplification when a mutation occurs in a primer binding site. ARMS has been used to study polymorphism of the apolipoprotein C I11 gene (LZO). Mitochondrial DNA (mtDNA) is inherited from a person’s mother. The association between mutations of mitochondial DNA and a variety of human diseases is having an impact on laboratory diagnosis of genetic disease. Methods for Lebeis hereditary optic neuropathy WON),the majority of cases caused by inheritance of mutations in mitochondrial DNA, have been developed. Mutation-specific PCR have been shown to be a rapid and inexpensive diagnostic method for LHON (L21). Solid-phase minisequencing has been used for the detection of point mutations in LHON W2). Other researchers have looked at deletions of mitochondrialDNA in dilated cardiomyopathy (L.231, showing a general association with deletions in mtDNA and advancing age. Pulse field gel electrophoresis and fluorescence in situ hybridization are techniques that have been used to investigate deletions and duplications of relatively large fragments of DNA (e.g., megabases). These methodologies have been applied to the quantitative measurement of duplicated DNA in Charcot-MarieTooth, a hereditary neuromuscular disease (U4, L.25). A simple method based on routine agarose gel electrophoresis and densitometric scanning has also been developed (LZ6). One of the most common hereditary disorders in Caucasians is deficiency of a-1-antitrypsin. Most cases involve either the Z or S mutations of the gene. A method for detection of the Z mutations based on colorimetric solid-phase minisequencing has been developed and evaluated, providing an accurate and simple alternative for the analysis of known point mutations (L27). Europium-labeled oligonucleotides have also been applied to the detection of the Z mutation (U8).The method, using timeresolved fluorescence, has been applied to Guthrie cards (dried blood spots on filter paper, also known as newborn screening cards). Mutational analysis of the gene associated with acute intermittent porphyria (AIP) offers a means of identifying affected patients. Molecular diagnosis of AIP has been extended to analysis of DNA extracted from hair roots (L29).PCR has been used to solve other mysteries in the clinical laboratory,including investigation of low serum lactate dehydrogenase in some individuals. Premature termination mutations have been shown to be responsible for lactate dehydrogenase H deficiency (DO). Multiplex PCR has Analytical Chemistry, Vol. 67, No. 72, June 15, 7995

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been applied to the detection of three common mutations of the hexosaminidase A gene that results in Tay-Sachs disease (&'I). A developing area of medical practice is in preimplantation genetics, the determination of the genetic makeup of an embryo before it is implanted, as in an in vitro fertilization program. In this area, the development and validation of laboratory procedures for the preimplantation diagnosis of Duchenne muscular dystrophy using a single cell has been reported (L32). Metabolic disorders usually diagnosed by conventional means have also moved into the realm of DNA diagnostics. A DNA method for the molecular diagnosis of maple syrup urine disease based on SSCP has been developed, enabling the rapid identification of mutations (L33). Previously unreported mutations in the phenylalanine hydroxylase gene that cause phenylketonuria (PKLJ) have also been identified (L34). Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a genetic disease that has been identified as a cause of some cases of sudden infant death syndrome. With dried blood spots as a sample, mutations in the MCAD gene have been identified using PCR and restriction enzyme digestion (L35).Molecular methods have been employed to study genetic determinants of nephropathy in type 1 diabetes (Us). This paper reports the use of denaturing gradient gel electrophoresis to study possible DNA sequence differences in the angiotensinogen and insulin receptor genes. DNA methods have been used to study differences in drug metabolism. SSCP has been used to detect differences in the human alcohol dehydrogenase and acetaldehyde dehydrogenase genes (L37).A DNA test that enables prediction of acetylation and debrisoquine hydroxylation phenotype has been developed

(L38). DNA ANALYSIS IN THE DIAGNOSIS AND MANAGEMENT OF CANCER Nucleic acid methods have become increasingly important in the diagnosis and management of the cancer patient. Rearrangements of DNA associated with leukemia and lymphoma are used as the foundation for the classificationof the cancer. Pulsed field gel electrophoresis was effective in the analysis of rearrangements of DNA involved in acute promyelocytic leukemia (L39). PCR has been used to detect fusion transcripts in leukemias with a translocation of chromosomes 4 and 11 (LAO). The ras oncogene is involved in the regulation of cell growth and differentiation.A rapid and sensitive method for detecting mutations of the ras oncogene in tumor samples has been reported (L41). An improved method for the measurement of another oncogene, c-erbB-2 has recently been reported (L42).In some tumors, including breast and ovary, there is an increase (amplification) in the number of copies of the c-erbB-2 oncogene (also known as HEE/neu), compared to normal tissue. The discovery of genes for several forms of inherited colon cancer has led to the development of testing strategies for at-risk family members. Genes associated with both familial adenomatous polyposis and hereditary nonpolyposis colon cancer have been identified (L4,L.43). One of the most important recent discoveries in genetic research was the identification of genes responsible for familial breast cancer, BRCAl (L44)and BRCA2 (LA5). Familial breast cancer account for approximately 5% of cases, primarily those where there is early onset of the disease. Identification of these genes on chromosome 17 (BRCA1) and 13 (BRCA2) make it 450R

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possible to detect at risk individuals using a DNA test. Currently, such a test is not available to the general population, owing to many of the concerns associated with presymptomatic genetic testing. A position statement regarding the use of DNA testing for cancer has been published (Us). DNA TYPING AND TRANSPLANTATION Bone marrow and organ transplantation remains an important area for DNA analysis. DNA testing has been used both in the selection of appropriate donors and for monitoring the progress of the transplant. A PCR-based method for amplification of Y-chromosome DNA to monitor the success of sex-mismatched bone marrow transplantation was devised (U7). Heteroduplex formation of PCR products has been used to screen for hisotocompatibility to match bone marrow donors and recipients (US). A single-step allele-specific PCR method has been developed for the HLA-DQ locus based on ARMS primers (US). A different method for HLA DNA typing has been reported using enzymelinked detection (WO).A 2-h test for the detection of HLA DQB and DQA alleles that uses sequence-specific primers was developed (Z.51). These methods have potential applications in the understanding of autoimmune diseases and the rapid matching of organ donor/recipient pairs. DNA ANALYSIS IN INFECTIOUS DISEASE DlAQNOSlS Providing rapid and sensitive methods for the identification of infectious agents is an important functionof the clinical laboratory. DNA probe methods are replacing and augmenting culture and antigen testing for a wide variety of microorganisms. A screening approach for fingerprinting of bacteria was reported (Z.52). This PCR method enables rapid and reliable fingerprinting of bacteria regardless of prior knowledge and is useful for identification and taxonomy. Direct DNA sequencing has been employed to provide analysis of heterogeneous viral populations (L.53). One of the important tasks associated with nucleic acid testing is isolation of DNA or RNA A one-step microbial DNA extraction method using Chelex 100 has been developed that is suitable for subsequent gene amplification (Z.54.This method has been tried clinically and was successful for the detection of human cytomegalovirus by PCR (W5). Growing areas of clinical interest for nucleic acids include the testing for hepatitis B and C virus. Direct and PCR-based methods for the detection of hepatitis B virus DNA were compared (Us). Other research has resulted in the development of a quantitative PCR assay for hepatitis B virus that utilizes colorimetric detection (Z.57). A microtiter plate assay for hepatitis B DNA has also been newly described (Z.58). Finally, a method for detection of the precore variant in the hepatitis B viral genome as been reported based on the amplification refractory mutation system (ARMS) (Z.59).The role of PCR in the diagnosis of hepatitis C virus (HCV) has been reviewed (L6O). The clinical utility of PCR RNA assays for hepatitis C virus was compared with HCV antibody assays such as enzyme immunoassay and immunoblot (L61).The quantitative detection of HCV RNA has been reported using a solid-phase signal amplification method (L62). A variety of methods for the isolation of HCV RNA from serum have been extensively evaluated (L63). With the reemergence of M. tuberculosis (MTB) as a major health care problem, the rapid identification of MTB is a desirable

goal for patient treatment and protection of health care workers. Current methods of ident5cation are typically culture dependent and can be lengthy because of slow growth of the organism. A commercial product for the direct analysis of MTB compared with PCR was evaluated (~564).Another PCR-based method for MTB in sputum has been reported (L65).The ligase chain reaction W R ) has been used in the development of several methods for MTB (L66,L67). An isothermal amplification method called strand displacement amplification (SDA) has been applied to MTB (L68). Recognition of multi-drug-resistant strains of MTB is becoming extremely important in a clinical context. A mixedlinker PCR method for rapid fingerprinting of MTB complex isolates has enabled classification of MTB strains (US).Proper control material is always a concern with clinical testing. This issue is addressed in a paper that describes a PCR method for production of internal control DNA in MTB assays (L70). Human immunodeficiency virus is an infectious agent that is not frequently detected by conventional molecular biology methods, unless amplified by PCR PCR for HIV can be used clinically to detect infection before seroconversion,coniirm infection where antigen testing is indeterminate, or monitor therapy in infected patients. PCR methods for HIV DNA or RNA include detection based on fluorescence (L71), colorimetry (1572,L73),electrophoresis (L74, or flow cytometry (L75). Conventionally, the clinical progression of HIV infection has been monitored by CD4+ lymphocyte counts. A new study shows that quantitative determination of HIV mRNA expression may be an independent predictor of disease progression (L76).Another research group has published a comparison of various DNA and antigen testing in a group of HIV-positive patients (L77). Mutations in the HIV genome have been studied, and a method based on mutation analysis may be useful in predicting resistance to certain forms of drug treatment (L78). A variety of other bacteria and viruses are of clinical importance. References for DNA methods for a handful of other infectious agents are of interest. Methods for cytomegalovirus has been reported based on PCR (L79)one including an internal control to allow for identification of false negative results (BO). A method to discriminatebetween integrated or episomal human papilloma virus in cervical samples has been developed (L81). Detection of cholera toxin by PCR has been compared with an immunosorbent assay, showing that PCR provides a sensitive and rapid assay for the diagnosis of cholera (L82).PCR direct antigen detection, and direct DNA hybridization were compared with culture techniques for detection of Chlamydia trachomatis (L83). A comparison of PCR and other diagnostic techniques for detection of helicobacter pylori infection in dyspeptic patients resulted in the conclusion that PCR had the best combined record of sensitivity and specificity (L84). DEVELOPING ANALYTICAL APPROACHES TO CLINICAL DNA TESTING Laboratorians interested in developing or refreshing a working understanding of DNA-based techniques are referred to several excellent reviews. A beginner’s guide to theory and practice of DNA-based techniques in clinical biochemistryis available (L85). A number of other general reviews of this area have also been published (126, L87). Applications and clinical experience of PCR in clinical laboratory medicine have been reviewed (L88,L89). The proceedings of the San Diego Conference, sponsored by the

American Association of Clinical Chemistry, also provide an excellent overview in this area (L90,L91). A review on the potential of PCR as a tool in pharmacokinetics provides an introduction into this developing area (1592).Selected recent advances in the area of immunoassays has been summarized, including DNA-based immunoassays (L93). Alternative amplification strategies that are different from PCR have also been reviewed. Ligation-based DNA diagnostics is the subject of a review (1594).An overview, and applications of the ligase chain reaction has also been published (L95). QB replicase as a method for amplifying nucleic acids has also been summarized in a review with over 60 references (L96).Standardization and control of DNA-based assays is an increasingly important issue. Appropriate standards and controls in DNA assays is the subject of a review (L.97).An interlaboratory statistical comparison of autoradiographic data is also available 6598). Developing rapid and efficient methods for isolation of DNA is important to further the implementation of nucleic acid diagnostics. An automated chromatographicprocedure for DNA from whole blood has been developed (L99). Guanidine salt is the basis for a simple method for extraction of RNA from cells and tissue (L100).Other work reports a method for isolation and evaluation of degradation of DNA from fixed tissue (L101).Triplehelix-mediated afEnity capture has been used as a method of rapid DNA isolation (L102). GENERAL METHODS AND IMPROVEMENTS IN PCR-BASEDTESTING A variety of methods have been developed or refined that enhance the general applicability of PCR to clinical diagnostics. PCR amplification of specific alleles (PASA) , a technique that can be used for the rapid, reproducible detection of point mutations or polymorphisms, has been reviewed (L103). Fluorescencebased image analysis has been applied to the detection of point mutations using PCR and single-strandconformational polymorphism electrophoresis (L104). Other work has focused on evaluation of a procedure for removal of DNA contamination using ultraviolet irradiation (L105).Three protocols for preventing false positives with PCR methods have been evaluated (L106). A general method for PCR amplification of regions with a very high G and C nucleotide content has been reported (L107).A novel approach to in situ amplification of DNA was developed, demonstrating the amplification of bacteria directly from tissue sections with a technique called localized in situ amplification (LISA) (L108).The ability to perform PCR in a silicon microstructure has been demonstrated,showing the potential to do amplifications in a format advantageous for minimization of consumables and amount of required sample, and applicable for point of care testing (L109). An automated closed-vessel PCR ampliication and detection system for use in in vitro diagnosticshas been evaluated (L110).It has been shown that large segments of double-stranded DNA can prime the PCR amplification of genomic DNA, demonstrating that oligonucleotide primers are not required for amplification (L111). METHODS FOR DETECTION OF AMPLIFIED NUCLEIC ACID PRODUCTS A triple-helical capture assay for quantification of polymerase chain reaction products has been developed. In this method, an oligonucleotide is used to form a triple-helical complex that can be easily detected owing to improved selectivity (L112).DNA Analytical Chemistry, Vol. 67, No. 12, June 15, 1995

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enzyme immunoassay @EM), a new colorimetric method based on an antibody that selectively recognizes double-stranded DNA was reported (L113). DEL4 has been applied to the detection of hepatitis virus as well as in the diagnosis of genetic disorders. PCR products have been quantjiied by a nonisotopic method that uses oligonucleotides covalently bound to microwells (L114). Time-resolved immunofluorometry has been applied to the determination of RNA sequences amplified by PCR showing an excellent limit of detection (L11.5).An immunochemical detection system was developed that utilizes an antibody to DNA-RNA hybrids, capable of detecting 10 amol of target DNA (L116). TECHNIQUES ON THE FRONTIER FOR POTENTIAL APPLICATION IN CLINICAL DIAGNOSTICS Fluorescence polarization (FP) has long been an important technology in the clinical laboratory. FP has been applied to the homogenous detection of nucleic acids, with a demonstrated detection limit of 1 fmol for human immunodeficiency virus (L117). A different DNA detection system has been demonstrated, based on coupling a serine protease (elastase) to a DNA probe, combined with a flourogenic Rhodamine substrate. This system demonstrates a detection limit of 2.46 fmol of conjugate (L118). Surface-enhanced Raman spectrometry has been used as a basis for detection of DNA hybridization. A DNA probe is labeled with a surface Raman-active material that can be detected when hybridization occurs (L119).The adsorption, immobilization, and hybridization of DNA onto a quartz crystal oscillator has been studied (L120). A sequence-selective biosensor for DNA that is based on electroactive hybridization indicators has demonstrated reusability (L121) and has been applied to the diagnosis of cystic fibrosis mutations (L122). In other work also focused on electrochemical detection of DNA, a gold electrode has been used for sequence-specific gene detection (L123). A related report describes the use of electrophore-labeled DNA probes (L124). An oligonucleotide was labeled with a compound that combines with a low-energy electron in the gas phase and can be detected by electron capture detection. The use of quadropole mass spectrometry has been applied to the detection of ionized duplex DNA fragments (L125). Time-of-flight mass spectrometry and matrix-assisted laser desorption has been used to detect a 40base oligonucleotide with 1-base resolution (L126). Fluorescence detection and sheath flow injection have been used to enable a size measurement of a single DNA molecule (L127). Native fluorescence detection of nucleic acids has also been demonstrated in capillary electrophoresis (L128). Other work in the area of electrophoresis includes the use of intercalating dyes to detect DNA fragments separated by capillary electrophoresis (L129). Improvements of DNA separation using pulsed field capillary electrophoresis have been demonstrated using dilute methyl cellulose solutions (L130). Several research groups focused on the improvement of DNAsequencing methods. A capillary electrophoresis sequencing method used two lasers and intensified diode array detection (L131). A fast manual, nonradioactive method for DNA sequencing, applicable for general laboratory use, has also been reported (L132). Two very different and exciting areas of DNA investigation on the horizon for clinical diagnosis are fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH). FISH involves the hybridization of labeled DNA probes to fixed 452R

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chromosomes on a slide. This enables the microscopic evaluation of whether hybridization has occurred and can be used for mapping and chromosome analysis (L133). CGH is based on FISH and involves the use of labeled total genomic DNA prepared from blood or tumor cells, mixed with normal differentiallylabeled DNA, The mixture is than hybridized with normal chromosomes that have been affted to slides. The DNA from the two sources will compete for complementary hybridization sites. If the DNA is essentially identical, all the chromosomes are viewed as the same color. If there is sequence differences between the DNA, there may be some regions where one color will predominate over the other. CGH has been used to detect complete and partial chromosome gains and losses, such as is observed in Down’s syndrome and leukemia (L134). Kristen J. Sko erboe received a B.S. in chemist j+om Colorado State Universityand a 5h.D. in analytical chemist fiom%ua State University. She ?s a dzploFate of the Amencan Board oYCltnica1 Chemtstty and also certified an Clinzcal Molecular Genetta by the Amencan Board ofMedical Genetacs. She as resently an assoczate laborato director at the Laboratoy of P a t h o k p a large clinical 1aboratory.ajiTated with Swedish Hospztal an Seattle, A. Her research znterests znclude the applzcatzon of unique s ectrqscopic and chromato ra hic. methods to clinical DNA analysts. d e r clznical interests incluk t i e dzagnosis of anherited forms of mental retardation and genetic hematologic disorders.

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