The Study Of Detecting β-thalassaemia Mutations From A Single Cell

Backgrounds and purposes: β-thalassaemia is one of autosomal genetic blood diseases characterized by absent or decreased production of normal beta hemoglobin. It is caused by the mutations within the beta globin gene. Over 160 types of mutations have been found in the whole world, and 21 of them have been oberserved among Chinese. It is one of the most common single disorders in South China. The frequency of β-thalassaemia trait (carrier status) is 0.67% in our country and as high as 6% in some provinces in South China.Approximately 8 different alleles comprise over 95% of the mutations found in China. Prenatal diagnosis for β-thalassaemia has been proven to be an effective strategy for controlling the incidence of new cases and is widely used in several countries where the disease is common. Successful preimplantation genetic diagnosis (PGD) protocols for beta-thalassaemia have been introduced using nested PCR followed by restriction fragment length polymorphism(RFLP), single-stranded conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE) and direct sequencing. The amounts of DNA for PGD and non-invasive prenatal diagnosis are very limited. The sensitivity of nested PCR is great enough to allow the analysis of DNA in a single cell. However, a single cell can be analyzed only once and it is difficult to detect mutltiple mutations of any one cell by nested PCR. Multiple copies of the DNA sequences present in a single cell are made by primer extention preamplication (PEP). PEP followed by nested PCR and reverse dot blot (RDB) have significant implications for detecting multipoint mutations in genetic disease diagnosis. The purposes of this study are as follows: to explore a technology for diagnosing β-thalassaemia multipoint mutations from a single cell simultaneously and to provide experimental evidences for the feasibility of applying PEP and DNA array technology in detecting multiple genetic mutations from a single cell.Methods: The research was divided into two parts: 1.Single lymphocyte was isolated under the inverted microscope equipped with micromanipulations. Two sorts of cell lysis methods (freezing-thawing and proteinase K lysis) were compared by their efficacy of multiplex amplification of DXZ1 and DYZ1 from a single cell. 2.A set of allele specific oligonucleotide (ASO) probes used for detecting the eight familiar β-thalassaemiamutations (CD41-42, IVS-Ⅱ-654, CD17, TATA box nt -28, CD71-72, TATA box nt -29, CD26, IVS-Ⅰ-5) were immobilized on a strip of nylon membrane. The genome of a individual cell was amplified by PEP with the mixture of 15-base random oligonucleotides. The aliquots (5μl) from PEP were used to amplify the objective gene fractions of β-thalassaemia gene by nested or semi-nested PCR. The membrane was hybridized with the final amplified products and then treated with Streptavidin - HRP and color development. Results:1. Under the inverted microscope equipped with micromanipulations, 80 lymphocytes were isolated. The amplification efficacy was 100% with cell lysis of proteinase K, and 80% with cell lysis of freezing-thawing. The amplification efficacy of two cell lysis methods showed significant difference (χ2=6.81,P<0.01).2. 3 lymphocytes were picked up from blood samples of 1 healthy female and 4 patients with known β-thalassaemia mutations respectively. Each single lymphocyte was lysed in the proteinase K buffer. The amplification efficacy was 93.3% and alle drop-out rate was 6.7%. RDB was applied to the final amplified products from the five participants. The results of diagnosis were same to the expected, and their genotypes were N/N, CD17(A→T)/N, IVS-Ⅱ-654(C→T)/CD17(A→T), CD41-42(-CTTT)/N and TATA box nt -28(A→G)/N, respectively.Conclusions:1.Proteinase K cell lysis method is simple and rapid. It can result in high amplification efficacy and meet to cell lysis and preparation of DNA template for amplification.2. A multiplex PCR assay to co-amplify DXZ1 and DYZ1 sequences from a single cell is a simple and accurate m...