Randall Keichi Saiki

HLA-DR, DO AND DP TYPING USING PCR AMPLIFICATION AND IMMOBILIZED PROBES

A simple, rapid, and precise method of typing HLA class II polymorphism would be valuable in the areas of disease susceptibility, tissue transplantation, individual identification and anthropological genetics. Here we describe a method of analysing class II sequence polymorphism based on polymerase chain reaction (PCR) amplification and hybridization with oligonucleotide probes. One valuable property of sequence‐based HLA typing strategies, like oligonucleotide probe hybridization, is that they reveal how and where two alleles differ, not simply that they can be operationally distinguished. The nature and location of HLA polymorphisms appears to be critical in disease association studies and are likely to be important in tissue typing for transplantation. New alleles at the DRB1, DPB1 and DQB1 loci are likely to be identified as this technology is applied to more and more samples, particularly in non‐Caucasian ethnic groups. A new allele is uncovered as an unusual pattern of probe binding and then confirmed by sequencing. This pattern is observed because class II polymorphism is localized to specific regions and virtually all ‘new’ alleles have polymorphisms in the region of probe binding. Obviously, any new allele with a new polymorphic sequence in a region for which typing probes are not available would not be revealed by oligonucleotide typing.

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.

Prenatal diagnosis of α-thalassemia by polymerase chain reaction and dual restriction enzyme analysis

SummaryAsian couples at risk for a fetus with homozygous α-thalassemia (hydrops fetalis) are often identified by their low erythrocyte mean corpuscular volume (MCV) and normal hemoglobin electrophoresis when little time remains to test their genotypes by restriction enzyme analysis. DNA analysis is performed directly on chorionic villi or amniocytes remaining after an aliquot is used to establish a backup cell culture. The polymerase chain reaction (PCR) protocol quickly determines whether the fetus has hydrops fetalis without waiting for cultured cells to grow. Previously, growing cultured fetal cells to obtain more fetal material to establish unambiguously the fetal genotype with two independent restriction enzyme digests absorbed a significant portion of the time remaining to complete prenatal diagnosis. A dual restriction enzyme digestion protocol was development using a 3′ζ-globin probe to clearly distinguish the most common α-thalassemia deletions that represent nearly all the α-thalassemia haplotypes in Southeast Asia.

Issues in implementing prenatal screening for cystic fibrosis: Results of a working conference

Purpose: To summarize a conference convened to examine how cystic fibrosis screening might appropriately be introduced into routine prenatal practice.Methods: Participants included experts from various relevant disciplines. Systematic reviews and data from individual trials were presented; issues were identified and discussed.Results: Judged by published criteria, prenatal cystic fibrosis screening is suitable for introduction. Screening can be performed cost-effectively by identifying racial/ethnic groups at sufficient risk and then using either of two models for delivering laboratory services. Validated educational materials exist. Ethical issues are not unique.Conclusions: Once adequate facilities for patient and provider education, testing, counseling, quality control, and monitoring are in place, individual programs can begin prenatal screening for cystic fibrosis.

[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.