CE UPDATE —MOLECULAR BIOLOGY 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 blot, 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 oligonucleotide 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 Southern blot, dot blot, and in situ hybridization. separating, or denaturing, the double-stranded DNA into individual strands, and then applying a marked nucleotide 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 enzyme. 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 gel electrophoresis, and o These double-stranded fragments can be sepa­ then isolated for further study. 0 rated by size using a technique known as agarose 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 nucleic acid 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 ethidium bromide, 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 ultraviolet 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 nucleotides 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 enzymes 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 restriction enzyme 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 Edwin Southern. 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 proteins. 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, protein 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 antibodies specific immobilized on the for the protein of interest. membrane surface, Northern blot 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.