W.G551D W.1717-1G-»A W.R553X W.C/T/?dele2,3 W.711+1G-»T W.3272-26A-»G W.3905insT W.R560T W.1898+1G-»A W.S1251N W.I148T W.3199del6 W.3120+1G-»A W.Q552X
Fig. 1 Schematic of multiplexed reversed line blot assay for cystic fibrosis. In an RLB, primers for amplification are synthesized using a 5'-biotin. These are incorporated in a multiplex PCR for most CFTR exons. Amplified exons are represented by double helices in the smaller test tube on the left. These are hybridized against short variant and normal oligonucleotide probes arrayed on solid supports, represented by the larger screw cap tube. The index for the Innogenetics CFTR19 LiPA RLB provides a guide to printed oligonucleotide probes. Subsequent to hybridization, the filters are extensively washed, blocked, and incubated with a horseradish peroxidase-conjugated streptavidin. For visualization, a synthetic substrate is added and the reaction is permitted to proceed for a short time. The resultant arrays shown here and labeled as a normal strip (N) and CF Carrier (heterozygous individual) are photographed and subjected to interpretation. Note that the normal strip evidences colorimetric indicators corresponding only to normal oligonucleotide probes, whereas the carrier displays a colorimetric reaction when probed with the M.F508del oligonucleotide (upper arrow on right). (RLB photos courtesy Donna Adams of Innogenetics and Dr. Amy E. Krafft of the Armed Forces Institute of Pathology.)
tested using this technology, often in less than 1 day. One prerequisite is that binding of oligonucleotides must be sequence-specific under uniform hybridization conditions. In most cases, the destabilizing effect of a single base-pair mismatch is sufficient to disrupt the formation of stable probe-target duplex. This specificity requirement can often be met either by adjusting the length, position, and strand specificity of the probe, or by varying the amount of probe applied to the membrane. Most probes are as short as is required to achieve specific hybridization, usually 15-23 base pairs in length, with a destabilizing mismatch toward the middle, but at least 3 base pairs from either end. The classic formula for predicting the dissociation temperature Td of an oligonucleotide, 2°C for each A/T and 4°C for each G/C, is simple but not necessarily accurate. In recent years, many algorithms have been developed that appear to give much better estimates and can assist in the design of probes for particular hybridization conditions. Some mismatches are less destabilizing than others. G/T mismatches are less destabilizing than C/A and these can be avoided by using probe designs on the opposite strand. Increased length provides for efficient competition with the secondary structure of the amplicon. Destabilizing mismatches are favored in RLB probe design. Trimethylammonium chloride (TMAC) is a quaternary ammonium salt that is used as an adjunct in hybridization reactions. Trimethyl-ammonium chloride eliminates the dependence of melting temperature on G-C content by reducing hydrogen bond energy between G-C base pairs. Additionally, TMAC binds specifically to A-T base pairs and increases their thermal stability. Thus the presence of TMAC at 3 M concentration in hybridization buffers reduces the melt temperature of an oligo with its complement to a function of length alone.
Reversed line blots are often arrayed to permit rapid interpretation of heterozygotes where one normal and one variant allele are indicated (Fig. 1). However, where two mutations are close to each other and fall within the sequence of the oligonucleotide probe (a situation often observed with respect to HbA, HbS, and HbC), a different pattern is obtained. DNA from individuals with HbS/HbS (sickle cell disease) will not hybridize to the normal probe at either the S or the C position. Instead, such DNA hybridizes solely to the HbS probe. This individual is distinguishable from HbS/HbC compound heterozygotes because the latter DNA will hybridize to both the mutant S and the mutant C probes. Similar RDB results are observed in the case of variants neighboring the AF508 CF mutation and those neighboring the IVS 1-1 and IVS 1-6 mutations, five nucleotides apart, that cause p-thalassemia.
In the past, RDBs have permitted the production of screening strips for aldolase B mutations (causing hereditary fructose intolerance),1-14-1 nondeletion a-thalas-semia, or adult onset mitochondrial disorders such as Leber hereditary optic neuropathy or hepatitis A contamination in food. A recently published reverse dot blot assay for congenital adrenal hyperplasia has high clinical utility in identifying sexually ambiguous newborns. These assays are flexible, inexpensive to implement, and use off-the-shelf commercially available hardware, reagents, and software. Both Tecan and Dynal manufacture specially engineered incubators and chemical dispenser devices that automate part of the RDB hybridization and developing process. While RDB assays are generally limited by an inability to detect large or quantitative deletions and an inability to characterize all but modestly expanded repeat sequences (ascertainment of the exact size of an expansion is often required for accurate molecular diagnosis), these strips can provide a means of accurate and reproducible genotype assignments. Automated spotting or line blotting of RLB strips allows the printing of large numbers of these with a minimum of operator intervention. Automation also permits higher density.
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