Establishment Of A PCD/LDR/Melting Curve Analysis For The Detection Of Thalassemia Mutations

BACKGROUND AND OBJECTIVEThalassemia is inherited as an autosomal recessive disorders, and is one of the most common single gene disorders in the world. Normal hemoglobin is composed of four protein chains, two a and two β globin chains arranged into a heterotetramer. Thalassemia patients produce a deficiency of either a or β globin cause by globin genes defects. This pathology is characterized by decreased Hb production and red blood cell (RBC) survival. The thalassemia syndrome is classified according to which of the globin chains, α or β, is affected. These2major groups, α-and β-thalassemia, are subclassified according to absent (α0and β0) or reduced (α+or β+) globin chain synthesis. In addition, where y-chains together with a-chains compose fetal hemoglobin (HbF) in the fetus and8chains in combination with a-chains compose hemoglobin A2in adults, impaired synthesis of y-globin or8-globin chains can occur. Because thalassemia heterozygosity confers some immunity against malaria, there is a particularly high incidence ofvthalassemia (2.5%-25%) in the Mediterranean basin, the Middle East, the tropical and subtropical regions of Africa, the Asian subcontinent, and Southeast Asia, where milder forms of the disease are most commonly seen.In China,the Provinces located in the south of the yangtze River have a relatively high incidence of thalassemia,especially Guangdong and Guangxi. According to statistics, the causative gene carriage ratio of α-and β-thalassemia about10%and2.8%.a-thalassemia is the result of deficient or absent synthesis of alpha globin chains,leading to excess beta globin chains. A-thalassemia is probably the most common monogenic gene disorder in the world. Although a-thalassemia most frequently results from deletion of one or both alpha genes from the chromosome, but the non-deletion α-thalassemia determinants may give rise to a more severe reduction in α-chain synthesis than the a deletion type of chromosome.In the present, there are lack of ideal treatment for a-thalassemia in clinic. The screen of a-thalassemia carrier, antenatal gene diagnosis and selective abortion is the only choice to control a-thalassemia.With the development of molecular biotechnology, various molecular diagnosis technologies have been applied to non-deletion thalassemia detection. SSCP, DGGE, TGGE technologies detect point mutations based on the change of mobility of mutated sequence in polyacrylamide gel electrophoresis caused by its altered structure. However, these methods are unfortunately time consuming, lacking sensitivity and technically difficult to implement. DHPLC proved to be a reliable, rapid, and highly sensitive methods for detecting point mutations. However, the samples are analyzed sequentially; thus, the major disadvantage with DHPLC is a relatively low throughput. Gene chips which has the advantages of large scale and high throughput offers a favored means to detect mutations of thalassemia,but they are expensive to create. DNA sequencing is considered as the "gold standard" for known/unknown mutation scanning, it still remains relatively expensive, laborious and time-consuming. At present,the major molecular biological method to diagnose non-deletion thalassemia is Reverse Dot Blot; however, complicated operation, false positive and flase negitive results makes it incompetence for screening. Ligase Detection Reaction (LDR) provides an elegant technique for mutations detection. LDR utilizes the ability of DNA ligase to preferentially seal adjacent oligonucleotides hybridized to target DNA in which there is perfect complementation at the nick junction. Utilization of thermostable Taq DNA Ligase enables repeated thermal cycles, resulting in a linear increase in ligation product. One of the most useful aspects of LDR is that sets of probes for a number of mutations can be multiplexed together, enabling several mutations to be detected simultaneously. At present, the technology that ligase detection reaction (LDR)coupled to a primary polymerase chain reaction (PCR) has a wide range of applications including scientific research and clinical diagnosis, especially in the field of nucleotide detection and mutation discrimination. A PCR/LDR assay amplifies the target sequence by PCR. and then detects the mutant signal by LDR.When coupled with an initial PCR amplification of the target sequence,LDR allows for high sensitivity detection of single base variants with extremely high specificity. In addition, attaching a fluorescent label to one of the ligation primers enables the detection of LDR products.The products may be seprarated by gel or capillary electrophoresis base on the different lentgh of LDR productions. If the other primer bears a unique zip-code complement,the ligation products can be detected by hybridization to a universal microarray spotted with the unique zip-codes.However, those post-LDR processes to separate the LDR products from the residual primers prior to detection are cumbersome, are the most time-consuming step in the entire assay procedure, making it difficult to quickly determine if mutated DNAs are present in samples. Several fluorescence resonance energy transfer (FRET)-based molecular probes, whose fluorescence signals change as a result of hybridization or enzymatic reactions, have been developed to enable separation-free detection of DNA. Molecular beacon is one of the molecular probes that set up based on FRET and the complementary pairing principles. These fluorescent molecular probes, which are very highly specific and sensitive, have now become one important tool in medical and biological researches.Since the PCR/LDR method can provide an distinguished tool for the detection of point mutations, we purpose to establish a rapid, highly sensitive and high throughput methods for detecting the mutations of thalassemia based on PCR/LDR method. The method can be used in rapid detecting test with highly accuracy and high throughput for the known mutations are needed for screening of this disease. MATERIAL AND METHODThe protocol of PCR/LDR/Melting curve analysis:first, amplified the target regions from genomic DNA of test samples by PCR; then.the amplicons were uesd as targets for LDR.When there were mutations existing, the LDR primers(upstream primer and downstream primer) were ligated by Taq DNA ligase.The LDR productions was formed the structure of molecular beacon and there was lower fluorescence intensity. During the melting curve analysis.the fluorescence intensity was increased as the rise of temperature since the structure of molecular beacon was broken at high temperature.Finally, the Tm values of molecular beacon that calculated by software were used to estimate the type of mutations.Methods1.Models:In this study,3most common mutations of a-thalassemia(Hb WS、Hb QS、Hb CS) were used as the models for establishing the PCR/LDR/Melting curve analysis.2.Designed of LDR primers:Totally3pairs of LDR primers were designed for the mutations.Each pair of LDR primer contains upstream primer and downstream primer.The upstream primer was designed to have stem sequence that was end labeled with HEX(Fluorophor) at its5’-end and a discriminating base at its3’-end;the downstream primer was labeled with BHQ1(Quencher)at its3’-end of a stem sequence and phosphorylated at its5’-end. The stem sequences of the primers were complementary to each other but not to the target.The molecular beacon models of LDR productions were designed and evaluated with the help of DNA folding software based on the work of the Zucker laboratory.3.Established the method with synthetic target and evaluated the specificity of Taq DNA ligase.4.The high-specific PCR amplification primers were designed according to the sequences of α2-globin gene,the region encompass the sites of all mutations of models.The amplicons were uesd as targets for establishing the single or multiplex LDR/Melting curve analysis and optimizating the reaction conditions.5.The data was analyzed by SPSS13.0software.RESULTS AND DISCUSSIONThe results of simulated experiment with aritificial targets show that the LDR/Melting curve analysis can test the point mutations with high specificity. And the Taq DNA ligase can work with high efficiency in different buffer.The ATm values (ATm is the difference between the predicted Tm value and actual Tm value) of3mutations were approximated.The results of single or multiplex LDR/Melting curve analysis with PCR productions were in conformity with the result of simulated experiments.CONCLUSIONSPCR/LDR/Melting curve analysis was proved to be a simple,highly accurate,and high throughput assay which can be used as a method for known point mutation of a-thalassemia. But one of the weaknesses of PCR/LDR/Melting curve analysis is that the method can ont discriminate homozygous mutation and heterozygous mutation.So PCR/LDR/Melting curve analysis was suitable for being the Preliminary screening in large-scale screen of thalassemia.