Nucleic acid hybridization is a process in which complementary single strands of nucleic acids combine to achieve a stable double-stranded nucleic acid molecule. This action has been utilized to establish a molecular or genetic relatedness between organisms and to characterize their genomes. Furthermore, this technique is one of several diagnostic tools useful for detecting a wide variety of conditions. Since the determination of the basic principles of duplex formation and stability in the 1950s, many variations of the hybridization techniques have been developed. Southern first transferred DNA fragments from agarose, after electrophoretic separation, onto nitrocellulose (1). The technique is known as Southern blotting. Alwine et al. (2) described shortly afterward a similar Northern blotting technique in which separated RNA strands are transferred from an agarose gel to a suitable solid support. A logical extension of these blotting techniques has been the dot or slot blot in which the sample is applied directly to the solid support without prior size separation (3). Over the years, these techniques have been further developed and modified extensively by many researchers across the world. The application of these methods is as varied as the procedures used (e.g., to determine the changes in the nutritional state of an environment, to establish taxa genetically, to distinguish pathogenic from nonpathogenic viruses, to analyze gene structure).
This chapter restricts itself to the application of Southern blotting to provide information relating to genetic diseases. The DNA sample, which can include blood, tissue biopsies, buccal scraps, amniotic fluid, cultured cells, and so forth, is generally digested using a restriction endonuclease and then subjected to electrophoresis in a horizontal agarose gel. After sufficient time has elapse to achieve adequate separation of the required fragments, the gel is soaked in an alkali solution to achieve denaturation of the double-stranded nucleic acid, then neutralized and prepared for transfer. Transfer to a nitrocellulose, polyvinylidene (PVDF), or Nylon membrane is achieved by a process of blotting in which buffer is drawn through the gel and the membrane. The fragments carried with the buffer are retained on the surface of the membrane. The retention is made more permanent through a fixation process. The blot can then be used in a hybridization with labeled probes to identify the fragments of interest.
There are many variations on this basic theme (4,5). Those procedures requiring the identification of fragments in excess of 10,000 bases advocate the use of a depurination step to improve the efficiency of large-fragment transfer. Methodologies using positively charged Nylon membranes often omit the neutralization step and advocate the use of alkaline transfer buffer, which can also serve as a fixative. There are many ways to achieve the transfer of DNA from the gel to the solid support. Southern's original method (1) describes the use of a capillary
From: Medical Biomethods Handbook Edited by: J. M. Walker and R. Rapley © Humana Press, Inc., Totowa, NJ
transfer procedure, and this remains the most widely used technique by far because of its low cost and convenience, transfer is often an overnight step (see Fig. 1). If speed is a requirement, the transfer process can be shortened by using specialized vacuum blotting apparatus or electroblotting devices. Such techniques allow transfer in 30-60 min compared to several hours for capillary transfer.
The membrane of choice is determined by the sensitivity required and the detection method to be used. The quantity of sample has a significant effect on both of these. The use of nitrocellulose usually results in low backgrounds and is recommended when the level of target is high. Nitrocellulose membrane is available in supported and unsupported forms, depending on the manufacturing method employed; however, the handling characteristics of the latter can be poor. Unsupported membranes are produced when the active substrate is cast as a pure sheet. Because of their fragile nature, unsupported membranes should be handled with care. Supported membranes are those for which the active substrate is cast onto an inert "web" or support.
Nitrocellulose membrane can bind 80-125 |g nucleic acid/cm2, which is significantly less than the binding capacity of 400-600 |g/cm2 for a Nylon membrane. Its ability to bind small molecules (<400 nts) is also poor, and transfer buffers must contain high salt concentrations to ensure efficient nucleic acid binding. Nylon membranes are available in uncharged and positively charged supported forms. Charged Nylon has a higher binding capacity and is particularly useful when working with low-molecular-weight nucleic acid. Binding to a Nylon membrane is independent of the ionic strength of the transfer buffer. However, backgrounds can be elevated. Where repeated use of a membrane in hybridization assays is needed, the use of Nylon membranes is strongly advised. Nylon is also recommended for use with medium- or low-abundance targets and is, in general, the membrane of choice when working with nucleic acids. PVDF membranes behave similarly to uncharged Nylon, but because of its hydrophobic nature, use in nucleic acid blotting is limited.
Factors Affecting Hybridization Rate and Stringency
How hybridization is affected
High temperature increases hybridization stringency; temperatures below Tm are recommended (for RNA long probe, 10-15°C below T • for DNA long probe, approx 25°C
Optimal hybridization in the presence of 1.5 M Na+; lowering ionic strength increases stringency.
Destabilizing agents (or Tm modifiers)
For example, formamide and urea; used to lower the effective hybridization temperature
Mismatched basepairs Duplex length
Mismatches lower hybridization rate
Hybridization rate increases with increased probe length
Increases rate of filter hybridization
Fixation bonds the target nucleic acid to the membrane. Suboptimal fixation will lower sensitivity by reducing target concentration and is particularly harmful if the blot is to be used more than once. The principal fixation methods of heat and ultraviolet (UV) light can be used with all types of membrane. Heat fixation is very reproducible but requires a vacuum oven for nitrocellulose. UV crosslinking, performed using an UV crosslinker (constant energy setting), is faster than heat. Alkali provides a third alternative method when charged Nylon membranes are used
Original methods describe the use of a DNA probe radioactively labelled by random priming or nick translation with [32P]dCTP to detect specific nucleic acid fragments immobilized on nitrocellulose membrane. Since then, many different methods for labeling nucleic acid probes ranging from short oligonucleotides to longer DNA or RNA fragments have been developed (6,7). Nonradioactive labeling kits and reagents are also available, finding favor in a number of niche areas (8). The role of the hybridization buffer is to provide conditions that promote hybridization between the labeled probe molecules and its complementary sequence immobilized on the membrane, and to simultaneously limit hybridization between sequences that are not perfectly matched (6). Table 1 lists factors affecting the hybridization rate and stringency (46). Many different formulations of hybridization buffers have been developed, containing inorganic salts and blocking agents such as Denhardt's solution (mixture of bovine serum albumin [BSA], Ficoll 400, and polyvinyl pyrrolidone), denatured DNA from salmon sperm (or other species), and heparin (4-6). A short prehybridization step in hybridization buffer is usually carried out to reduce nonspecific background hybridization before adding the labeled probe. This is especially important in genomic Southern blots that contain all genomic sequences on the membrane. Hybridization with the probe is usually carried out for several hours to allow hybridization between low-abundance sequences. Although various rapid-hybridization buffers containing volume excluders are available to speed up this step. After hybridization, the blot is washed to remove unhybridized probe. The stringency of washing is usually controlled by stepwise reductions in the ionic strength of wash buffer and/or by temperature (4-6). The replacement of the old plastic bag technology with specialized temperature-controlled rotisserie devices for performing hybridization and washes has resulted in more consistent results and safer handing of radioactivity. After washing, the blot is subjected to autoradiography with X-ray film to visualize the bound probe (9).
2. Southern Blotting in the Diagnosis of Human Disease
Southern blotting has been applied to the diagnosis of many human diseases at the molecular level. These genetic diseases are caused by point mutations, gene rearrangements, or the amplification of genes or specific sequences within the genome. These methods have in common that restriction-digested genomic DNA is size-separated in agarose gel electrophoresis, transferred onto the membrane, and hybridized with gene-specific probes.
Restriction fragment length polymorphism (RFLP) analysis was one of the early methods to diagnose point mutations implicated in genetic diseases (see Chapter 3). This method was based on the observation that if a point mutation changes a restriction fragment recognition sequence, it is possible to detect this change by Southern blotting analysis in which the affected restriction enzyme is used to cut genomic DNA prior to analysis. The change in the size of detected fragments with a gene-specific probe signals the presence of mutation in the analyzed gene. For example, this method has been applied to the diagnosis of hemophilia A, which is the most common inherited bleeding disorder, affecting approx 1 in 5000 males worldwide. Hemophilia A is an X-linked, recessively inherited bleeding disorder that results from a deficiency of procoagulant factor VIII (FVIII). Affected males suffer from joint and muscle bleeds and easy bruising, the severity of which is closely correlated with the level of activity of coagulation factor VIII (FVIII:C) in their blood. Gitschier et al. demonstrated using Southern blotting that it was possible to diagnose the disease in 42% of affected families (10). Having identified the BclI polymorphism, X-linked inheritance was demonstrated in three generations of a Utah family. DNA from a family member was restricted with BclI, electro-phoresed on a 0.8% agarose gel, and blotted on to Nylon membrane and probe with a complementary sequence within the factor VIII gene labeled with radioactivity. Twelve bands were observed by autoradiography. Eleven hybridizing bands remained constant, and one varied in position at either approx 0.9 or 1.1 kb in size.
The presence of gene point mutations has also been diagnosed with Southern blotting using allele-specific probes. These are usually short oligonucleotides in which the mismatched sequence is situated in the middle. By carefully controlling the hybridization and wash conditions (temperature and ionic strength of wash buffers), it is possible to distinguish between the binding of oligonucleotides differing by only one nucleotide. This method has been applied, for example, to distinguish between the normal human P-globin gene and the sickle cell P-globin gene (11,12). Sickle cell anemia is caused by a single base change, resulting in the replacement of glutamic acid residue at position 6 of the protein (hemoglobin) by a valine residue.
Gene rearrangements can be diagnosed with Southern blotting if a probe hybridizing to the affected areas is used. Rearrangement is detected by observing change in the size and pattern of hybridized genomic restriction fragments. This type of analysis has been applied to the diagnosis of acute promyelocytic leukemia (APL), a subtype of the cancer acute myeloid leukemia. The disease is characterized by abnormal, heavily granulated promyelocytes, a form of white blood cells. APL results in the accumulation of these atypical promyelocytes in the bone marrow and peripheral blood, and they replace normal blood cells. At the molecular level, the disease involves translocation between the retinoic acid receptor-a (RAR-a) on chromosome 17 and the promyelocytic leukemia locus (PML) on chromosome 15. This results in the transcription of novel fusion messenger RNAs. By separating restriction-digested genomic DNA in pulse field gel electrophoresis followed by hybridization with probes derived from PML and RAR-a genes, it was possible to detect translocation events that correlated with disease progression (13).
Gene amplifications are implicated in many diseases. Charcot-Marie-Tooth (CMT) syndrome is a common autosomal-dominant neuromuscular disorder. The disease is characterized by a slowly progressive degeneration of the muscles in the foot, lower leg, hand, and forearm and a mild loss of sensation in the limbs, fingers, and toes. The first sign of CMT is generally a high arched foot or gait disturbances. Other symptoms of the disorder may include foot bone abnormalities such as high arches and hammer toes, problems with hand function and balance, occasional lower leg and forearm muscle cramping, loss of some normal reflexes, occasional partial sight and/or hearing loss, and, in some patients, scoliosis (curvature of the spine). Genetically, the disorder is usually characterized by duplication of a region on chromosome 17 through unequal crossover. As a result, affected patients carry three copies of this region. One diagnostic approach to CMT, type 1A exploits Southern blot hybridization and the relative intensity for three polymorphic MspI RFLP bands within the duplicated area to judge whether patients have two or three copies of this region using a region-specific probe. In order to normalize the observed intensity of the signal resulting from the CMT gene probe, another probe derived from unconnected sequences is used (14).
The most significant changes in the use of Southern blotting in diagnosis have been seen since the introduction of primer-specific polymerase chain reaction (PCR) and automated non-radioactive sequencing techniques. Mutation and gene deletions once detected via Southern blot analysis are now routinely analyzed with these rapid and inexpensive methods, which are often fully automated. Cystic fibrosis, Duchenne muscular dystrophy, sickle cell anemia and thalassaemia, to name a few, are now diagnosed by polymerase chain reaction (PCR). PCR methods can be completed in as little as 4 h, whereas Southern blotting can take up to 5 d to achieve the same result. More important, the amount of DNA required for analysis is significantly less with PCR amplification methods. The Southern blotting diagnosis method generally requires 5-10 ^g of genomic DNA. The introduction of a PCR-based method is generally only achieved once the gene defect has been characterized at the molecular level. Hence, research into disorders where the defect is unknown or further information is required still utilizes Southern blot analysis as an important research tool. In the routine laboratory, the use of Southern blotting is restricted to those diseases that require additional information the Southern blot can provide. One such disease is Fragile X; however prescreening using PCR analysis is common.
Fragile X syndrome is a common genetic disease. This inherited form of mental retardation affects 1 in 4000 males and 1 in 8000 females (15). Males with fragile X often exhibit characteristic physical features and accompanying autistic and attention-deficit behaviors. Individual IQs are in the range 35-70 (16-18). Approximately 30% of females with full mutations are mentally retarded, and their level of retardation is, on average, less severe than that seen in males.
In 1943, Martin and Bell (19) were able to link the cognitive disorder to an unidentified mode of X-linked inheritance. In 1967, Lubs (20) discovered excessive genetic material extending beyond the low arm of the X chromosome in affected males. Diagnosis was originally based on cytogenetic analysis of metaphase spreads, but less than 60% of the affected cells in affected individuals showed a positive result. With this variability in the test, the carrier status of individuals could not be determined. Interpretation of the result is further complicated by the presence of other fragile sites in the same region of the X chromosome.
The fragile X gene (FMR1) is located in chromosomal band Xq27.3 and encodes an RNA-binding protein, which was initially characterized in 1991 (21-23) and contains a tandemly repeated trinucleotide sequence (CGG) end at its 5' end. The disease is caused by the absence of a functional FMR1 gene product (24). A small number of individuals classified as fragile X cytogentically have expansion at the nearby FRAXE locus, which also contains an unstable CGG repeat (25-28). The normal distribution of the repeat in the unaffected population varies from 6 to 50. Affected individuals are classified into one of two major groups; premutations of approx 50-200 repeats and full mutations with more than 200 repeats. Some alleles with approx 45-55 copies of the repeat are unstable and expand from generation to generation; others are stably inherited (29-31).The larger the size of the female premutation, the greater the risk of expansion during meiosis (32). Individual male or females carrying a premutation are unaffected (20,29,30). Males pass on the mutation relatively unchanged to all their daughters, all of whom are unaffected.
In some cases of premutation, an accurate estimate of the size of the expansion is required, most especially in family studies. This increase in resolution is achieved by Southern blot assay, using Pstl digestion of genomic DNA and detection with a probe close to the repeat array. Characteristic of the full mutation is methylation of the promotor region of the gene (CpG island) (33); this correlates directly to gene inactivation. This is an important event in the disease pathogenesis, its effect on clinical severity is, however, unpredictable, especially in females.
Today, two main approaches are used in diagnosis of fragile X syndrome: PCR (36-39) and Southern blot analysis (40). The use of flanking primers allows the amplification of the region of DNA containing the repeats. The size of the PCR product is therefore indicative of the number of repeats present. However, the efficiency of the PCR reactions inversely relates to the number of repeats; hence, large mutations are more difficult to amplify and can fail to yield a PCR product. False negatives by PCR can also be an issue caused by the presence of normal and full mutations in some male patients (41). No information as to the extent of methylation can be determine by a PCR-based assay. Southern blotting allows both the size of the repeat segment and methylation status to be assayed simultaneously. Methylation-sensitive restriction enzymes such as Eagl or Nrul can be used to distinguish between methylated and unmethylated alleles. When combined with EcoRl, these enzymes give fragment sizes of 2.6 kb from normal unmethylated FMR-I genes. Methylated normal genes are not cut by these enzymes and yield 5.1-kb EcoRl fragments. Methylated and unmethylated expansions are indicated by the presence of bands or smears above the 5.1-kb and 2.6-kb fragments, respectively.
lt is common practice in diagnosis laboratories to use PCR for prescreening and only to proceed to Southern blotting for those samples that fail to amplify (males) or show a single normal allele (females). lf the etiology of mental impairment is unknown, DNA analysis for fragile X syndrome should be performed as part of a comprehensive genetic evaluation, which includes routine cytogenetic analysis. Cytogenetic studies are critical because constitutional chromosome abnormalities have been identified as frequent or more frequently than fragile X mutations in mentally retarded individuals referred for fragile X testing. For individuals who are at risk because of an established family history of fragile X syndrome, DNA testing alone is sufficient. lf the diagnosis of the affected relative was based on previous cytogenetic testing for fragile X syndrome, then it is advised that at least one affected relative should be included in the DNA testing profile. Prenatal testing (42) of a fetus is indicated following a positive carrier test in the mother. When the mother is a known carrier, DNA testing can be offered to determine whether the foetus inherited the normal or mutant FMR1 gene. Results must be interpreted with caution because the methylation status of the FMR1 gene is often not yet established in chorionic villi at the time of sampling. Follow-up amniocentesis might be necessary to resolve an ambiguous result. ln a very small number of patients, deletions or point mutations (43-48) rather than trinucleotide expansion are responsible for the syndrome. ln these cases, linkage (31,49,50) or rare mutation studies are more useful.
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