Amplification of a GC-rich sequence from barley by a two-step polymerase chain reaction in glycerol

PCR amplification of GC-rich sequences may fail due to poor denaturation or secondary structures that block elongation. Successful amplification of a 672-bp sequence encoding the barley α-amylase/subtilisin inhibitor (69% GC) was achieved in a simple two-step procedure with the addition of 20% glycerol and a high annealing temperature. This protocol may be useful for the amplification of other GC-rich sequences.     

In vitro effects of organic solvents on immunity indicators in serum

Serum treatment in vitro with organic solvents (chloroform, ether, toluene) failed to produce an effect on immunoglobulin levels and activity. After chloroform and ether treatment, no complement activity could be determined, with chloroform-treated serum beginning to express anticomplement activity against autologous, allogenic and xenogenic sera. The classical pathway of complement activation (C1, C4, C2, C3) was primarily inhibited, whereas the alternative pathway remained unaffected. Chloroform-treated sera exhibited significantly declined levels of C1-INH, C3 and C4 as well as of circulating immune complexes. Toluene did not influence any of the parameters tested, while ether blocked complement activity without affecting either the concentration or activity of the other components under investigation. The obtained findings are discussed from the aspect of organic solvent applications in preparing immune products and determining immunity indicators in the serum or other biological fluids.     

Inhibitors of the polymerase chain reaction

The information on the applied aspects of the polymerase chain reaction (PCR) is updated. In particular, the main inhibitor of PCR, considerably decreasing the sensitivity of the method both at the lysis stage of the tested material and due to the degradation of the DNA matrix and primers and/or to the direct inhibition of the activity of DNA polymerase, are described. The compounds, most frequently distorting the course of the reaction while testing clinical blood samples, bioptic samples, sputum, etc., are characterized. Testing concrete clinical material with the use of PCR was shown to require differentiated approach both at the stage of choosing the adequate method for the preparation of samples and at all other stages, including, e.g., the corresponding DNA polymerases or at the stage of heating for decreasing endonuclease activity or for IgG denaturation. Information on the causes of false negative results of PCR and the variants of their elimination, useful under practical laboratory conditions, is given.     

Estimation of the reaction efficiency in polymerase chain reaction

Polymerase chain reaction (PCR) is largely used in molecular biology for increasing the copy number of a specific DNA fragment. The succession of 20 replication cycles makes it possible to multiply the quantity of the fragment of interest by a factor of 1 million. The PCR technique has revolutionized genomics research. Several quantification methodologies are available to determine the DNA replication efficiency of the reaction which is the probability of replication of a DNA molecule at a replication cycle. We elaborate a quantification procedure based on the exponential phase and the early saturation phase of PCR. The reaction efficiency is supposed to be constant in the exponential phase, and decreasing in the saturation phase. We propose to model the PCR amplification process by a branching process which starts as a Galton-Watson branching process followed by a size-dependent process. Using this stochastic modelling and the conditional least-squares estimation method, we infer the reaction efficiency from a single PCR trajectory.     

聚合酶链反应在牛血清支原体检测中的应用

为了探讨聚合酶链反应在牛血清支原体检测上的应用价值,以支原体高度保守的rRNA操纵子(支原体基因组中16SrRNA的编码区序列)设计引物,采用碱裂解法提取牛血清中支原体DNA作为模板进行聚合酶链反应。结果表明,阳性、阴性和内控对照都扩增出了预期的条带,聚合酶链反应与支原体培养法比较,有灵敏、快速、特异性高的特点,可用于牛血清中支原体的常规检测。     

Diagnosis of haemophilia B using the polymerase chain reaction

The polymerase chain reaction (PCR) was used to amplify specific DNA sequences within the factor IX gene of haemophilia B patients and their relatives. Three of the amplified fragments contain polymorphic sites, which can be used as markers in segregation analyses. These restriction fragment length polymorphisms (RFLPs) were until recently detected by Southern blotting after digestion with the restriction enzymes Taq I, Dde I and Xmn I. All three RFLP's are located in introns of the factor IX gene and together are informative in approximately 70% of all cases. Each of the polymorphisms was successfully used in carrier detection studies after amplification of the relevant fragments. This method is also suitable for rapid antenatal diagnosis. Additionally we were able to amplify all eight exons of the factor IX gene including the splice junctions and a part of the 5'-region. Large deletions or insertions can be detected without further analysis. Several possibilities for the rapid detection of point mutations after DNA amplification have been described recently. The complete amplification of all functional parts of the Factor IX gene in combination with these new techniques should enable us to detect the majority of mutations leading to haemophilia B.