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Home , Blot (biology), Dot blot, Edwin Southern, Northern blot, Southern blot

CE UPDATE — II

James Wisecarver, MD, PhD

Techniques Used To Test Native DNA Downloaded from https://academic.oup.com/labmed/article/28/2/121/2503736 by guest on 29 September 2021

The first article of this series described the key structural features of DNA and how it is extracted ABSTRACT Several techniques commonly are used to test from cells for study. After a DNA sequence has native high-molecular-weight DNA obtained from cells and been extracted from cells and purified, the genetic tissues, including the Southern , dot and slot blot proce­ information contained within the sequence can be dures, and in situ hybridization of tissue sections with examined through a variety of techniques. This article discusses the techniques commonly probes. These techniques work well when used to test native high-molecular-weight DNA there is an adequate amount of fresh tissue or cells available to obtained from cells and tissues, including provide a source for the intact DNA. These techniques involve , , and in situ hybridization. separating, or denaturing, the double-stranded DNA into individual strands, and then applying a marked Southern Blot The technique known as Southern blot analysis is probe and allowing it to hybridize to a complementary DNA used widely for analyzing the size of certain DNA sequence that may be present. These techniques are used to fragments. The sequence of interest is prepared determine whether a particular DNA sequence is present. by digesting intact DNA collected from a patient's This is the second article in a three-part series on DNA. Other articles discuss the struc­ C cells with a restriction endonuclease . 0 tural properties of DNA, how it is extracted from cells for study, some of the basic tools '£ Following complete digestion, a collection of II used to gain useful clinical information, and DNA amplification techniques. On comple­ o double-stranded DNA fragments remains, rang­ 'E tion of this series, readers will be able to describe the composition of DNA, how the 3 ing in size from a few hundred to several thou­ strands are arranged, how to extract DNA from tissues prior to testing, and how DNA £ £ sand base pairs. fragments are prepared by enzyme digestion, separated using , and o These double-stranded fragments can be sepa­ then isolated for further study. 0 rated by size using a technique known as gel electrophoresis. This technique involves plac­ After the DNA fragments have been separated, From the ing the fragmented DNA into wells in a slab of the double-stranded fragments are denatured Department of Pathology and agarose gel and submersing the gel in a buffer into single strands by soaking the gel in sodium Microbiology, © chamber in the electrophoresis apparatus. hydroxide followed by neutralization. These DNA University of c 0 Electric voltage is applied, causing the negatively pieces then are transferred onto a piece of elec­ Nebraska Medical ti charged fragments to migrate toward trostatically charged paper or filter membrane. In Center, Omaha. ( the positively charged region, or anode, of the gel the original description of this technique, the slab Reprint requests apparatus. The gel matrix acts as a sieve, allowing of gel was placed in a tray of buffer, and the filter to Dr Wisecarver, the smaller fragments to move through the gel material was placed on top of the agarose gel. Department of Pathology and matrix easily while the progressively larger frag­ Absorbent paper toweling was placed on top of Microbiology, ments have more difficulty migrating through the the filter material allowing the buffer to work University of gel. These DNA fragments are invisible to the upward through the gel and the filter membrane Nebraska Medical naked eye but can be visualized if the gel is into the absorbent toweling (Fig 1). The migra­ Center, 600 S 42nd stained with a dye such as , tion of buffer also transfers the DNA fragments St, Omaha, NE 68198-3135. which binds to the DNA. After staining, the gel from the gel onto the membrane surface. Most can be placed on an light source. laboratories use vacuum- or air pressure-blotting Orange fluorescence indicates the presence of the devices that greatly shorten the time necessary to DNA within the lanes of the gel. transfer the DNA onto the membrane. The single-stranded DNA fragments that have been Downloaded from https://academic.oup.com/labmed/article-abstract/28/2/121/2503736 by guest on 28 May 2018 FEBRUARY 1997 VOLUME 28, NUMBER 2 LABORATORY MEDICINE 121 transferred are immobilized permanently onto sequence. The appropriate conditions for each the membrane surface by brief exposure to ultra­ procedure must be determined. When using a violet light. commercially prepared kit, care should be taken The next step is to determine the location and during probing and washing steps to adhere to size of the particular DNA sequence of interest. the times and temperatures outlined in the An oligonucleotide probe with a base sequence instructions to obtain accurate results. that is complementary to the sequence of interest Following these wash steps, the membrane is prepared containing a label to permit detection. should be exposed to x-ray film for a period of One simple technique for labeling probes is to hours to days, depending on the strength of the Test Your incorporate radioactive as the probe is signal emitted from the probe. If radioactive

Knowledge assembled. The location of the radioactive probe probes are used, no further steps are needed. Downloaded from https://academic.oup.com/labmed/article/28/2/121/2503736 by guest on 29 September 2021 Look for the CE can be detected by exposing the membrane to x- When using enzyme-labeled probes such as alka­ Update exam on ray film. More recently, nonisotopic techniques line phosphatase, however, a substrate must be Molecular Biology have been developed to label probes with added to the membrane surface before exposure to (702) in the March such as alkaline phosphatase. A suitable enzyme issue of Laboratory the film. This substrate, when cleaved by the alka­ Medicine. Participants substrate can be used to detect the presence and line phosphatase enzyme, emits photons of light. will earn 3 CMLE location of the bound probe. These light photons act similarly to the particles credit hours. The membrane blot is placed in a salt solution released from decay of a radioactively labeled containing the labeled probe at a temperature probe and alter the silver present in the x-ray film. that will permit the probe to hybridize, or bind, Development of the x-ray film following exposure to its complementary DNA fragment bound to to the membrane surface yields an image that will the membrane. During the incubation period, disclose the location of the DNA fragments con­ Fig 1. In the which often is several hours, the probe eventually taining the sequence of interest (Fig 2). Southern blot proce­ dure, DNA fragments will find the complementary base pair sequence To determine the size of this fragment, a size that have been pre­ on the membrane and bind to this target DNA. marker is placed in the gel and is transferred to pared by cutting Following hybridization, the solution containing the membrane. By comparing the size of fragments native DNA with a the labeled probe is removed, and the membrane detected by the probe with the size marker, it is is washed several times with a salt-buffer solution possible to estimate the number of bases present in are separated in to remove any unbound probe from its surface. agarose gel through the fragment to which the probe hybridized. electrophoresis. The By adjusting the salt concentration and the Most laboratories use a 1-kilobase DNA ladder in fragments are trans­ temperature, one can carefully remove the non- which the sizing bands are approximately 1,000 ferred onto a nylon specifically bound probe, being careful not to dis­ bases apart. membrane by plac­ rupt the bonds between the probe and the ing the gel in a intended target sequence. This careful considera­ buffer bath and plac­ Northern and Western Blots ing the membrane tion of the ionic strength and temperature used The Southern blot was named for its inventor, Dr on top of the gel. during the wash steps often is referred to as the . Other investigators began to use Paper toweling is "stringency" of the procedure. If the temperature this technique to separate and identify RNA frag­ placed on top of the is too low or the ionic strength is too high, the ments and . Investigators using this tech­ membrane, drawing probe might stick to the membrane in a nonspe­ the buffer upward nique to isolate messenger RNA (mRNA) dubbed from the sponge cific fashion. If the temperature is too high, the it northern blotting, as opposed to Southern blot­ located in the pan, probe might separate from the intended target ting, which examines DNA fragments. Not to be through the gel and outdone, chemists membrane, and into used the term western the toweling. As this The Southern Blot Procedure occurs, the DNA blotting to describe a tech­ fragments leave the nique in which proteins gel and are deposited Buffer wicks up through are separated electro- on the membrane membrane into paper toweling phoretically, transferred surface, producing a Paper towels Filter membrane to membranes, and iden­ "blot." After this ^4, A transfer is complete, tified through the use of the fragments are labeled specific immobilized on the for the protein of interest. membrane surface, analysis of and a labeled probe mRNA is more technically is added to localize Gel containing Buffer in tray Sponge soaked in demanding than similar the DNA fragments separated DNA buffer supporting gel containing the fragments analyses of DNA. RNA is sequence of interest.

Downloaded from https://academic.oup.com/labmed/article-abstract/28/2/121/2503736 by guest on 28 May 1220182 LABORATORY MEDICINE VOLUME 28. NUMBER 2 FEBRUARY 1997 degraded easily, because numerous Fig 2. An autoradio­ Autoradiograph From Southern Blot graph is prepared by ribonuclease enzymes present in exposing x-ray film mammalian cells quickly break to a membrane blot down these molecules. Investigators that has been performing such studies must be hybridized with a careful to treat all reagents with a labeled probe. The visible bands on the substance such as diethylpyrocar- X-ray film __ film correspond to bonate (DEP-C), which inactivates the location where these ribonucleases so that the RNA the labeled probe has become bound sequence they are trying to detect is Dark bands indicate sites where the labeled (hybridized) to the

not degraded. The Southern, north­ Downloaded from https://academic.oup.com/labmed/article/28/2/121/2503736 by guest on 29 September 2021 probe hybridized to the membrane. The tra­ ern, and procedures DNA fragment being sought. ditional approach are similar in that large macromole- has been to incorpo­ cules are separated on a membrane, rate radioactive The size of the DNA fragments nucleotides into the followed by detection of a particular can be estimated by comparing sequence of interest or protein mol­ them with the DNA marker ladder probes. Recently, that was run on the same gel. nonradioactive ecule using an oligonucleotide labels have been probe or marker system, developed that use respectively. In Situ Hybridization enzyme-labeled probes and sub­ The previous sections describe techniques in strates that release Dot/Slot Blot Methods which DNA, removed from cells and tissues, is photons of light If you wish to determine whether a particular separated and placed on artificial substrates and when the enzyme nucleotide sequence is present in a patient's sam­ then probed for sequences of interest. In some acts on it. These ple, shortcuts are available. In this analysis, cases, it is preferable to apply these labeled probes light photons or enzyme digestion and subsequent electrophoretic directly to cells and tissues to localize the source products of radio­ active decay pro­ separation need not be performed. Instead, the of the signal. This technique is known as in situ duce dark bands DNA sample is rendered single-stranded by alka­ hybridization. Using this method, the DNA with­ on the film after it line denaturation or heat, spotted onto a nylon in the native tissues is denatured by heating, fol­ is developed. membrane, and then probed using an oligonu­ lowed by application of a labeled probe that cleotide probe that was labeled in the same way as binds to the complementary sequence of interest in c in the Southern blot technique. This procedure is (Fig 4). Following several wash steps, a detection 0 known as a "dot blot." For example, a probe spe­ method is used to localize the signal that indi­ n u cific for a particular mutation (eg, sickle cell ane­ cates areas in which the probe has bound to the e mia), can be used to test DNA from the patient's tissues. In cases in which radioactive probes are 3 cells to determine whether the mutation is present £ used, the tissue sections on microscope slides are £ (Fig 3). In this example, the probe is designed to dipped in a silver emulsion similar to an x-ray o o detect the thymine substitution for adenine, film. These slides are kept in the dark for a period which results in the incorporation of valine at of time (from days to several weeks), after which position 6 in the (3 hemoglobin chain. The the emulsion is developed in a fashion similar to hybridization stringency conditions are adjusted developing film. so that the probe will bind if thymine is present, but will not bind if adenine is present. This approach The Dot Blot Technique can detect individuals who have this i mutation owing to the presence of a Patient DNA is spotted and linked to membrane substrate. positive signal when the blots are ooooOOOOOo' OOOOO Labeled probe is exposed to film. In many cases, het- ooooo added to membrane ooooo and allowed to erozygotes and homozygotes can be ooooo hybridize. Fig 3. The dot blot distinguished due to the increased technique involves signal intensity generated when two spotting DNA onto a membrane substrate defective copies of the gene are pre­ Blot is washed to remove and then using a excess labeled probe. sent, as in persons with sickle cell Blot is exposed to x-ray labeled complemen­ OOOOO film. Dark, exposed areas disease (homozygotes). ooooo»ooo correspond to samples where tary nucleic acid oooto probe has become bound, probe to determine ooooo indicating that the target ooooo sequence is present. whether a specific sequence is present in the specimen.

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In some cases, a fluorescent marker is attached This guide can help you better understand the techniques commonly to the probe. These probes then are used in a used to test native high-molecular-weight DNA. technique known as fluorescence in situ Electrophoresis—A method for separating macromolecules on the hybridization (FISH) to detect sequences on indi­ basis of size and net electrical charge. vidual . Such techniques are useful for detecting extra copies of a with­ Dot blot—A technique to determine whether a particular nucleotide in cells (eg, trisomy 21 in Down syndrome). sequence is present in a patient's sample. A DNA sample is rendered single-stranded by alkaline denaturation or heat, spotted onto a Conclusion nylon membrane, and then probed using an oligonucleotide probe. DNA fragments that have been prepared by cut­

Heterozygous—Possessing different copies of a gene from each parent. ting DNA using bacterial restriction enzymes can Downloaded from https://academic.oup.com/labmed/article/28/2/121/2503736 by guest on 29 September 2021 Homozygous—Possessing identical copies of a gene from each parent. be transferred onto a solid supporting membrane using Southern blotting. After the fragments have Hybridization—Binding of two complementary base sequences. been transferred to the membrane, a labeled In situ hybridization—A technique in which DNA within the native nucleic acid probe, having a sequence comple­ tissues is denatured by heating, followed by application of a labeled mentary to the target sequence, is used to pin­ probe that binds to the complementary sequence of interest. point the location of the target sequence on the Oligonucleotide probe—A string of nucleotides used to detect the membrane blot. Slot or dot blot techniques can presence of a complementary nucleic acid sequence. be used to detect the presence or absence of a tar­ get sequence within a mixture of unseparated Slot blot—The same procedure as dot blot (see above); the differ­ DNA pieces, without having to follow the steps of ence is the shape of the template used to spot the DNA onto the electrophoretic separation. membrane. In situ hybridization is used to identify a target Southern blot—A technique to separate, identify, and determine the nucleic acid sequence within tissue sections. This size of DNA fragments. process is similar to immunohistochemistry tech­ Western blot—A technique in which proteins are separated elec- niques used in many laboratories. The difference trophoretically, transferred to membranes, and identified through the is that in situ hybridization incorporates a DNA use of labeled antibodies specific for the protein of interest. denaturation step to separate the cellular DNA into individual strands. A labeled nucleic acid Alternatively, these probes can be labeled with probe then is used to detect the presence of the an enzyme similar to those used for blotting tech­ target nucleic acid strand. In situ hybridization is niques. Following probe hybridization, the particularly useful for detecting viral nucleic enzyme substrate is placed on the slide, and a acids within tissues.® color reaction develops at the site of probe local­ ization. This process is similar to immunohisto- Selected Readings chemical detection of antigens, which is used Darnell J, Lodish H, Baltimore D. Molecular . New York, NY: Scientific American Books; 1990. routinely in many pathology laboratories. In situ Kirby LT. DNA Fingerprinting: An Introduction. New York, techniques have been used for detecting viral NY: Stockton Press; 1990. nucleic acid sequences (eg, human papillo- Lewin B. The extraordinary power of DNA technology. In: Lewin B, ed. Genes V. Oxford, England: University Press; Fig 4. In the tech- mavirus) within infected cells. 1994:633-656. nique known as in Piper MA, Unger ER. situ hybridization, Nucleic Acid Probes: A Primer for Pathologists. Chicago, 111: tissue sections are In Situ Hybridization ASCP Press; 1989. mounted on slides, Ross DW. Tools of recombi­ and the DNA in the nant DNA technology. In: section is denatured Labeled probe hybridized Ross DW, ed. Introduction to to tissue by heating. A labeled Tissue section Molecular Medicine. New probe is hybridized York, NY: Springer-Verlag; onto the section. The 1992:29-52. presence of the bound probe can be detected by expos­ ing the slides to a film emulsion or by adding the appropri­ Microscope slide ate substrate if an enzyme-labeled probe is used.

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