Ernest S. Kawasaki

Genetic Analysis Using Polymerase Chain Reaction-Amplified DNA and Immobilized Oligonucleotide Probes: Reverse Dot-Blot Typing

The reverse dot-blot method is a simple and rapid diagnostic procedure that allows screening of sample for a variety of mutations/polymorphisms in a single hybridization reaction. Several methods of immobilizing the oligonucleotide probes are discussed. The reverse dot-blot method has several unique properties that are valuable in a diagnostic setting: (1) the typing results from a single sample can be located on a single strip. This facilitates scanning and interpretation of the probe reactivity patterns and minimizes the potential for user error. (2) The test can utilize premade typing strips. This minimizes user labor as well as error potential and allows the use of standardized reagents. (3) Unlike dot-blot/oligonucleotide typing, only the PCR product is labeled, eliminating the potential problem of probes labeled to different specific activities. This method has already been used in the areas of forensic genetic typing (the HLA-DQ alpha Amplitype test), tissue typing for transplantation (the HLA-DR beta) test, cystic fibrosis screening, as well as in a variety of research applications.

Bladder cancer. Human leukocyte antigen II, interleukin-6, and interleukin-6 receptor expression determined by the polymerase chain reaction

The prediction of tumor biology rarely rests upon a single characteristic of the malignancy. The analysis of a single gene can complement standard histologic evaluation. The investigation of new parameters as well as the routine clinical analysis of gene expression is often limited because of the small amount of tissue available. This is particularly true of de novo human bladder cancers because they are generally small or handled in such a way as to hinder the analysis of multiple different parameters. Analysis of expressed mRNA by the polymerase chain reaction (RNA/PCR) is a method that allows the development of a profile of bladder cancer gene expression. The authors report the use of the RNA/PCR method to examine in bladder cancer the expression of the human leukocyte antigen (HLA) class II gene family (HLA‐DR, DQ, and DP) as well as interleukin‐6 (IL‐6) and the interleukin‐6 receptor (IL‐6R). All de novo transitional cell carcinomas, one squamous carcinoma, and two transitional cell carcinoma cell lines expressed the majority of HLA class II genes. All samples expressed IL‐6R RNA whereas production of IL‐6 message was limited to one of the cell lines and to the high‐grade bladder cancers. These results were combined with stage, grade, and DNA content to develop a profile of the cancers examined. Although an improved predictive index based on gene expression analysis by RNA/PCR has not been realized, a broader survey of human tumors for expression of these genes and others is likely to refine the classification of bladder cancer.

[44] – Genetic Analysis Using Polymerase Chain Reaction-Amplified DNA and Immobilized Oligonucleotide Probes: Reverse Dot-Blot Typing

Publisher Summary The difficulty of analyzing single-copy genes in genomic DNA has been overcome by use of the polymerase chain reaction (PCR) to first amplify the target sequence to high abundance, followed by hybridization with the allele-specific oligonucleotide probe. This chapter discusses the analysis of PCR-amplified DNA with sequence-specific oligonucleotide hybridization probes. The conventional dot-blot method in conjunction with PCR amplification and oligonucleotide probes has greatly simplified the analysis of any DNA or RNA sequence, including those involved in genetic diseases, HLA polymorphisms, cancer, and so on. If the sample size is large, the PCR/dot-blot method is convenient to use, especially when the number of probes required is small. However, as the number of probes required for genetic typing increases, this method becomes cumbersome because the PCR product must be immobilized on a number of membranes, each of which is hybridized to a different labeled oligonucleotide probe. To alleviate this difficulty, a method known as the “reverse dot blot” is used. In this technique, the allele- or gene-specific oligonucleotide probes are bound to the filter instead of the PCR products, and the amplified DNA labeled during the PCR is used to hybridize to the immobilized array of probes.