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CE U P D A T E — M O L E C U L A R BIOLOGY III James Wisecarver, MD, PhD Amplification of DNA Sequences The techniques for studying intact, high-molecular-weight DNA obtained from tissues, discussed earlier in this series, require DNA with ample numbers of target sequences. In some instances, however, only a small amount of material might be available for study (eg, a few cells from a fineneedle aspirate) or only a few target sequences available for detection (eg, a few viral particles in a sample of cerebrospinal fluid). In these cases, it is useful to selectively amplify the DNA target sequence of interest, thereby providing ample copies of the DNA sequence for study. This article will discuss methods for producing multiple copies of DNA sequences and techniques for detecting variations in nucleotide sequences, including DNA sequence analysis. ABSTRACT \This article discusses some of the tools available to amplify DNA sequences. These techniques are extremely useful for detecting the presence of small amounts of a DNA target sequence. They also can he used to detect short sequences in DNA that has degraded partially. The cloning procedure, useful for producing many copies of a sequence, also is described. Methods for comparing nucleotide sequences within amplified sequences are discussed, including heteroduplex analysis and single-strand conformational polymorphism (SSCP) analysis. Methods for performing DNA sequence analysis are presented as well. This is the final article in a three-part series on DNA. Other articles discussed the structural properties of DNA, how it is extracted from cells for study, some of the basic tools used to gain useful clinical information, and the techniques commonly DMA Amplification Techniques used to test native high-molecular-weight DNA obtained from cells and tissues. On o The probe hybridization technology mentioned earlier requires high-molecular-weight DNA from a large number of cells. Each cell contains only one copy of the DNA. Oligonucleotide probes require that many copies of the target DNA be present to provide enough hybridization points to permit target localization on a blot. In similar fashion, in-situ hybridization works best when multiple viral or other target sequences are present within each cell, again providing a sufficient number of sites for probe hybridization to permit detection of the signal. In other instances, however, only a few copies of the target sequence may be present. In such cases, it is desirable to amplify this limited amount of target sequence so that it can be detected and studied. Strategies recently have been developed to amplify a single target sequence millions of times so that it can be detected more easily. The most widely used amplification strategy is the polymerase chain reaction (PCR). To use this procedure, the target sequence to be amplified must be completion of this series, readers will be able to describe the composition of DNA, c how the strands are arranged, how to extract DNA from tissues prior to testing, and how DNA fragments are prepared by enzyme digestion, separated using gel elec- u known. Typically, this target sequence ranges from 100 to 1,000 base pairs in length. To perform PCR, two small oligonucleotide primer sequences must be synthesized that typically are 16 to 20 base pairs in length. These primer sequences are complementary to the 3' ends of the sequence to be amplified (Fig 1). Initially, the DNA is denatured by heating in the presence of the synthesized primer sequences. As the reaction mixture cools, some of the DNA strands reanneal with their original complementary partner strands. Because the synthetic primer pieces are present in excess numbers, however, they are more likely to bind to the complementary region on the native DNA strand. With the primers bound to the native DNA, the polymerization (DNA synthesis) reaction is free to proceed. The technologic breakthrough that permitted the automation of this technique was the discovery of a VOLUME 28, NUMBER 3 E E 0 trophoresis, and then isolated for further study. MARCH 1997 3 From the Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha. 6 I Reprint requests to Dr Wisecarver, Department of Pathology and Microbiology, University of Nebraska Medical Center, 600 S 42nd St, Omaha, NE 68198-3135. LABORATORY MEDICINE 191 The Polymerase Chain Reaction DNA sequence to be amplified 1 Denaturation Primers bind to complementary regions. 1 N e w DNA strands are synthesized. 3' y Newly synthesized D N A \ 3' 5' 1 5' 3' Now two copies of sequence serve as template. \ 5' 3' 5' Primer binding 5J 5' 1 3' 5; 3' 5' 3' 3' 5' \ 3' 5' DNA synthesis 5' 5' 3' 3' 5' 5' After two cycles, four copies of original sequence are available to serve as templates for synthesis of additional strands during the next cycle. Fig 1. Polymerase chain reaction. After determining a sequence of interest, short, 15-to-20 base-pair primer sequences are synthesized that are complementary to the 3' end of each strand. After the native, doublestranded DNA has been denatured, the primer strands bind to the denatured strands and serve as a start point for DNA synthesis. The reaction is catalyzed by a thermostable DNA polymerase enzyme that is resistant to the high temperatures needed to denature the newly synthesized strands so that the second cycle of the reaction can occur. 192 DNA polymerase enzyme from a strain of bacteria collected from a mineral hot spring. These bacteria are capable of surviving at high temperatures, and their biologic enzymes are capable of withstanding temperature extremes. Consequently, this enzyme does not lose its ability to synthesize new DNA despite numerous denaturation steps. After the primer sequences have attached, or annealed, to the target DNA strand, the DNA polymerase enzyme will generate new DNA strands by incorporating individual nucleotide bases that are provided in the reaction mixture (Fig 1). After synthesis of the new DNA strands, the reaction mixture again is heated to 96°C to denature the newly generated DNA pieces from the original DNA strands. These new DNA strands, along with the original strands, can serve as templates for additional amplification cycles. If these steps are repeated 25 to 30 times, it is possible to amplify the original DNA segment 1 million times. Millions of copies of the sequence can be generated within a few hours. As outlined above, this amplification process provides large numbers of copies of the original target sequence. Often, it is necessary only to LABORATORY MEDICINE VOLUME 28, NUMBER 3 MARCH 1997 determine whether amplification occurred. For example, you can detect viral DNA from a blood sample using a pair of primers specific for a short sequence within the viral genome that is specific to the virus and is not found in mammalian cells. Following amplification, the presence of viral DNA would yield a PCR product of the appropriate size; whole samples not containing viral DNA would have no product. These amplified products can be detected simply by placing the PCR reaction mixture in agarose gel and performing electrophoresis. Because the length of the DNA target sequence is known, the presence of a distinct band in the agarose gel following ethidium bromide staining that is of the appropriate size is evidence that viral DNA was present in the sample. To further confirm that this is viral DNA, the amplified fragments can be transferred to a membrane using the Southern blot procedure, and an oligonucleotide probe, specific for a sequence within the viral PCR product, is applied to confirm that the amplified product is virus DNA rather than nonspecific amplification of human DNA. In the case described, the presence of a band is evidence that the viral sequence was present in the specimen. The absence of a band is problematic, however, in that one cannot be certain whether the sequence indeed was absent, or that the reaction conditions were inappropriate for the PCR reaction to proceed. In most cases, a positive control reaction is used as well, in which a second set of primers to a normal human gene certain to be present are added to ensure that the PCR conditions were adequate and that all reagents were added. Techniques based on the PCR are being applied to clinical situations to detect both human and microbial genes within tissues and blood specimens from patients. These amplification techniques can be performed relatively quickly and require only a small amount of DNA. The Southern blot procedure requires DNA from many cells and up to several weeks to perform. The PCR technique, using rapid and simplified DNA isolation procedures, can be performed in only a few hours using DNA from a small number of cells. Potentially, results can be available the same day the patient's sample is received in the laboratory. Also, the increased sensitivity of amplification procedures allows for the detection of a very small number of copies of a target sequence in a relatively large specimen (eg, a few copies of a viral sequence in a sample of cerebrospinal fluid). DNA Cloning Gene Cloning One of the more important advances in molecular biology has been the ability to remove segments of mammalian DNA and insert them into small circular portions of bacterial plasmid DNA. This technique involves removing the DNA sequence of interest by cutting it away from the intact genomic DNA using restriction enzymes. This DNA restriction fragment is inserted into a circular piece of plasmid DNA for insertion into bacteria (Fig 2). The bacteria then are propagated in culture using a system that is selective for the bacterial organisms containing the plasmid DNA. This often is achieved using a plasmid vector containing an antibiotic resistance gene. The bacteria then are cultured in the presence of the antibiotic. Only bacteria containing the plasmid with the gene to inactivate the antibiotic are capable of sustained growth within the medium. Using this approach, large numbers of copies of the DNA target fragment can be generated as the bacteria multiply. The human DNA fragments can be isolated by lysing the bacteria using a special buffer, collecting the plasmid DNA, and then excising the human portion using the original restriction enzyme. The DNA sequence of the human fragment can be determined as outlined below. Using bacterial plasmids, only small portions of human DNA can be inserted. For larger sequences, other cloning carriers (vectors) have been developed (eg, yeast artificial chromosomes). D N A Sequencing Sometimes, it is desirable to know the entire nucleotide sequence of a particular portion of DNA. This is especially useful when looking for point mutations (single nucleotide changes) within a gene encoding a biologically important protein. Sequence analysis requires a large number of copies of the particular sequence of interest. These copies can be obtained either by cloning the sequence into a plasmid vector, inserting the vector into competent bacteria and amplifying it, or by using PCR to produce large numbers of copies of the target sequence. After the sequence has been amplified, a primer is synthesized that will hybridize at the 3' end of the DNA strand to be sequenced. Individual nucleotides now are added along with a DNA polymerase enzyme. In addition, one of the four bases (ie, adenine, thymidine, cytosine, or guanine) is labeled with a radioactive marker. A small amount of a single nucleotide is added as a Human DNA (Double stranded) Circular bacterial plasmid DNA (Double stranded) Treat with restriction enzyme Plasmid ring is opened. DNA containing region of interest is cut out. Human DNA Circular plasmid DNA containing human insert di-deoxyribose derivative. Four separate reactions are set up, each containing a different labeled base and di-deoxyribose terminator. The advantage of using these di-deoxynucleotides is that there is no hydroxyl group at the 3' position to permit chain extension. Wherever one of these labeled bases is incorporated, the growing nucleotide strand is terminated. By providing an excess of unlabeled single deoxy bases, the chances of incorporating the di-deoxy chain terminators is a random event. A large number of molecules will incorporate the regular deoxy nucleotides and the strand extension will continue. Occasionally the di-deoxy terminator will be incorporated, resulting in chain termination. The four reaction mixtures then are separated electrophoretically in an acrylamide gel with each lane representing a different labeled base. Using the appropriate acrylamide concentration, it is possible to separate the individual nucleotide fragments on the basis of size, each being one base longer than the previous fragment. Using this method, it is possible to read off the sequence directly from the gel. Automated methods have been developed that incorporate fluorescent labels into the di-deoxy MARCH 1997 VOLUME 28, NUMBER 3 Fig 2. DNA cloning strategy. A fragment of interest is isolated from human DNA using restriction enzymes. The excised fragment is inserted into the circular plasmid DNA vector that has been cut open using the same restriction enzyme used to prepare the human fragment. The plasmid DNA containing the human insert is then incorporated into bacteria, usually Escherichia coli, where it is copied as the bacteria replicate. Using this approach, large numbers of copies of the human DNA insert can be obtained. LABORATORY MEDICINE 193 Fig 3. Single-strand conformational polymorphism. Following denaturation, as the nucleic acid strands are allowed to cool, they assume a threedimensional structure based on internal areas of complementary sequence, and consequently fold up into hairpinlike structures. These differences in threedimensional conformation affect the migration of the DNA fragments in acrylamide gel. Two polymerase chain reaction products that have an identical migration pattern are assumed to be identical, while products that migrate differently have areas where the sequences differ. Single-Strand Conformational Polymorphism Product A I Polymerase chain reaction products: double-stranded DNA Product B Heat to denature. As mixture cools, the individual strands fold to form hairpin-like structures based on internal areas of base complementarity. I t e r m i n a t o r s . Using this approach, each chain t e r m i n a t o r can be labeled with a different color and all of the reactions can be run in a single lane on a gel. A laser scans the gel, determining which color terminator is p r e s e n t at each l o c a t i o n . Using this method, the software within the instrument is able to d e t e r m i n e the sequence with a high degree of accuracy. Single-Strand Conformational Polymorphism Single-strand conformational p o l y m o r p h i s m (SSCP) is Products migrate differently based used to detect subtle variaon alterations in three-dimensional tions in short DNA sequences structure. such as PCR-amplified products. This technique typically is used to compare sequences from two or m o r e individuals to d e t e r m i n e whether they are identical or whether a mutation has occurred. To perform this type of analysis, the amplified products are denatured into single strands and then separated using acrylamide gel The following guide will help you in your study of DNA amplification electrophoresis. Following denaturation, as the septechniques. arate strands cool, they fold up on themselves, assuming a three-dimensional conformation based Amplification—A process to produce multiple copies of a specific DNA on intrastrand regions of base complementarity. If sequence. there are focal base differences within the same Di-deoxyribose derivative—A ribose sugar in which hydrogen replaces the sequence s e g m e n t from two individuals, or hydroxyl groups at both the 2' and 3' positions on the ring. between two different allelic forms of gene from Gene cloning- -A method for producing quantities of a specific DNA the same individual, these strands fold up in sequence. slightly different conformation due to these complementarity differences. These conformational Heteroduplex analysis—A modification of single-strand conformational differences can be seen when the strands are seppolymorphism that can be used to determine sequence differences in allelic arated using acrylamide gel electrophoresis (Fig variants of a gene. 3). Single-strand conformational polymorphism Nucleotide sequencing—A method to determine the entire nucleotide has been used to detect m u t a t i o n s in normal sequence of a particular portion of DNA. Sequence analysis requires a large human genes such as the p53 tumor suppressor number of copies of the particular sequence of interest. gene. Plasmid vector—A piece of circular DNA contained within bacterial organA modification of SSCP can be used to deterisms. Under appropriate conditions, this plasmid can be introduced into bacmine sequence differences in allelic variants of a terial organisms, bringing with it additional genetic information. gene. This technique is called heteroduplex analyPolymerase chain reaction (PCR)—A strategy that uses synthesized oligonusis and involves amplifying the genetic locus of cleotide primers to amplify a single target DNA sequence so that it can be interest using PCR. The amplified products are detected and studied more easily. m i x e d t o g e t h e r , d e n a t u r e d by h e a t i n g , a n d allowed to cool. In some cases, the original two Single-strand conformational polymorphism—A technique used to detect strands from each amplicon reanneal to their subtle differences in nucleotide sequences; typically used to compare sequences from two or more individuals to determine whether they are idencomplementary partner (homoduplex). In other tical or whether a mutation has occurred. instances, the strand from one allele binds to a A Guide to Amplification Terminology 194 LABORATORY MEDICINE VOLUME 28. NUMBER 3 MARCH 1997 strand from the other nonidentical allele (heteroduplex). While there is a high degree of sequence complementarity, the match is not perfect, leading to a short region or regions that are not bound, thereby altering the electrophoretic migration. By analyzing the reaction mixture using an acrylamide gel, it is possible to separate these heteroduplexes from the homoduplexes, indicating that some areas of noncomplementary sequence are present and therefore that these pieces of sequence are not identical. Potential Uses The techniques outlined in this series have a wide range of clinical uses within the laboratory. The most obvious and exciting applications are in the area of clinical microbiology. Using sensitive amplification-based strategies, small amounts of microbial DNA can be detected in clinical samples. These techniques also have wide applicability to hematopathology. The Southern blot techniques and, more recently, PCR-based assays have been used to detect clonal rearrangement of immunoglobulin and T-cell receptor genes, indicating the presence of a clonal proliferation of lymphocytes. Detection of a clonal expansion of lymphoid cells suggests the presence of a lymphoid neoplasm. Other PCR techniques have been developed to detect chromosomal translocations typical of other lymphoid and hematopoietic malignancies, as well as some types of solid organ neoplasms. Innis MA, Gelfand DH, Sninsky JJ, White TJ. PCR Protocols: A Guide to Methods and Applications. San Diego, Calif: Academic Press; 1990. Piper MA, Unger ER. Nucleic Acid Probes: A Primer for Pathologists. Chicago, 111: ASCP Press; 1989. Ross DW. Polymerase chain reaction. Arch Pathol Lab Med. 1990; 114:627. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989. Please let us know your opinion of t h e Molecular Biology (702) series. 1. T h e series m e t t h e objectives stated in t h e abstract. Deficient 1 Excellent 5 Test Time Look for the CE Update exam on Molecular Biology (702) following this article. Participants will earn 3 CMLE credit hours. 2. T h e series p r o v i d e d useful technical data or o r i g i n a l ideas. Deficient 1 Excellent 5 3. T h e i n f o r m a t i o n p r o v i d e d in t h e series w a s new and timely. Deficient 1 Excellent 5 4. Technical p o i n t s w e r e explained clearly a n d w e r e easy t o c o m p r e h e n d . Deficient 1 Excellent 5 5. T h e text w a s organized logically. Deficient 1 2 3 4 Excellent 5 6. Illustrations, charts, a n d tables helped explain text a n d a d d e d t o series value. Deficient 1 2 3 4 Excellent 5 C o m m e n t s : (attach a d d i t i o n a l pages, if necessary) 0 e 3 Summary An increasing number of molecular tests are being developed, and the results are beginning to appear in the literature monthly. Virtually all tests are based on exploiting the physical and chemical properties of DNA using techniques such as DNA amplification, gene cloning, and nucleotide sequencing. An understanding of the basic concepts of nucleic acid structure and physical properties will help the reader evaluate these reports for possible clinical significance. Eventually, these techniques will find their way into most clinical laboratories. A basic understanding of these concepts will aid the pathologist or technologist in analyzing the results and troubleshooting the test procedures.© Selected Readings and Reference Materials E E o 0 T h a n k y o u f o r y o u r input. M a i l this f o r m w i t h y o u r e x a m or alone t o : Laboratory Medicine, 2100 W Harrison St, Chicago, IL 60612-3798. c .2 'o © Correction In the first article of this series, titled "The ABCs of DNA" {Lab Med. 1997;28:48-52), the CH groups in Fig 1 on page 49 should be hydroxyl (OH) groups. The corrected figure follows. 5" OH —CH 2 Base V 4" OH 3" OH Y 5" OH—CH 2 Base r 4" OH 3" H 2' Deoxyribose Brown TA. DNA sequencing: The basics. Oxford, England: Ribose Oxford University Press; 1994. Glick BR, Pasternack JJ. Molecular Biotechnology: Principles and Applications of Recombinant DNA. Washington, DC: ASM Fig 1. Structural comparison of ribose and deoxyribose. The only difference in Press; 1994. structure is the exchange of a hydrogen molecule for the hydroxyl group attached to the carbon molecule in the 2' position. MARCH 1997 VOLUME 28, NUMBER 3 LABORATORY MEDICINE 195 E 0