A PCR method for uniform amplification of a mixture of DNA templates differing in GC content is described using the two enzyme approach (Klentaql and P fu DNA polymerase) and a combination of DMSO and betaine. This method was applied to amplify the CGG repeat region from the fragile X region.Development of PCR for the amplification of nucleic acids has revolutionalized several fields of molecular biology. Since the introduction of this method several parameters of the system have been modified to amplify longer nucleic acid fragments (Foord and Rose 1994, and references within). A variety of DNA polymerases have been used for PCR because of their improved thermal stability (Lawyer et al. 1993) and editing functions (Lundberg et al. 1991;Mattila et al. 1991).Use of a two-enzyme approach (Klentaq I and Pfi~ DNA polymerase) has enabled the amplification of fragments up to 35 kb in length (Barnes 1994;Cheng et al. 1994).Several reagents have been shown to facilitate DNA strand separation either because they disrupt base pairing [e.g., dimethylsulfoxide (DMSO), formamide] or isostabilize DNA [e.g., tetraalkylammonium (TAA) salts such as tetramethylammonium chloride (TMAC), tetraethylammonium chloride, and N,N,N-trimethylglycine (betaine)] (Melchior and yon Hippel 1973; Rees et al. 1993). Some of these reagents have been included in PCR reactions to amplify GCrich templates. Recently, it has been shown that at lower concentrations TMAC can enhance the amplification of AT-rich DNA fragments, and at higher concentrations it can inhibit enzyme amplification (Chevet et al. 1995). However, templates with high GC contents are variably amplifiable or completely unamplifiable even with the addition of DMSO, formamide, glycerol, TMAC, or 7-deazaguanosine triphosphate (7cdGTP), which lowers the melting temperature of the substituted DNA (McConlogue et al. 1988;Bookstein et al. 1990;Hung et al. 1990;Sarkar et al. 1990;Pomp and Medrano 1991). In spite of all the advances described above there is no single method that has been developed that could amplify to a similar extent all DNA templates in a heterogeneous mixture.There are a number of PCR applications (e.g., construction of cDNA library from a small number of cells, representational differential analysis of two different genomic DNA, or cDNAs from two different cell types) for which it would be highly desirable to have a method whereby a mixture of DNA fragments of similar length (0.5-10 kb) can be amplified with equal efficiency regardless of base composition. Therefore, we explored amplification by using isostabilizing quarternary amines that did not inhibit the DNA polymerases. Betaine is a natural amino acid analog and has many useful properties such as osmoprotection of Escherichia coli cells, stabilization of proteins against thermal denaturation, and isostabilization of DNA (Csonka 1989;Santoro et al. 1992; Rees et al. 1993). However, use of betaine as a reagent for amplification of GC-rich templates has not been explored.In this report we studied the efficiency of ...
The mechanism of interaction of O-amino-D-serine (OADS) with sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) (SHMT) was established by measuring changes in the enzyme activity, absorption spectra, circular dichroism (CD) spectra, and stopped-flow spectrophotometry. OADS was a reversible noncompetitive inhibitor (Ki = 1.8 microM) when serine was the varied substrate. The first step in the interaction of OADS with the enzyme was the disruption of enzyme-Schiff base, characterized by the rapid disappearance of absorbance at 425 nm (6.5 X 10(3) M-1 s-1) and CD intensity at 430 nm. Concomitantly, there was a rapid increase in absorbance and CD intensity at 390 nm. The spectral properties of this intermediate enabled its identification as pyridoxal 5'-phosphate (PLP). These changes were followed by a slow unimolecular step (2 X 10(-3) s-1) leading to the formation of PLP-OADS oxime, which was confirmed by its absorbance and fluorescence spectra and retention time on high-performance liquid chromatography. The PLP-OADS oxime was displaced from the enzyme by the addition of PLP as evidenced by the restoration of complete enzyme activity as well as by the spectral properties. The unique feature of the mechanism proposed for the interaction of OADS with sheep liver SHMT was the formation of PLP as an intermediate.
The interaction of aminooxy compounds such as aminooxyacetate (AAA), L-canaline, and hydroxylamine with sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) was studied by absorption spectra and stopped-flow spectrophotometry and compared with the unique feature of interaction of O-amino-D-serine (OADS) with the enzyme [Baskaran, N., Prakash, V., Appu Rao, A. G., Radhakrishnan, A. N., Savithri, H. S., & Appaji Rao, N. (1989) Biochemistry (preceding paper in this issue)]. The reaction of AAA (0.5 mM) with the Schiff base of the enzyme resulted in the formation of pyridoxal 5'-phosphate (PLP) and was biphasic with rate constants of 191 and 19 s-1. The formation of the PLP-AAA oxime measured by decrease in absorbance at 388 nm on interaction of AAA with the enzyme had a rate constant of 5.2 M-1 s-1. On the other hand, the reaction of L-canaline with the enzyme was slower as measured by the disruption of enzyme-Schiff base than the reaction of OADS and AAA. In contrast, the formation of PLP as an intermediate could not be detected upon the interaction of hydroxylamine with the enzyme. The reaction of D-cycloserine with the enzyme was much slower (1.6 x 10(2) M-1 s-1) than the aminooxy compounds. These observations indicate that the aminooxy compounds that are structural analogues of serine (OADS, AAA, and canaline) formed PLP as an intermediate prior to the formation of oxime, whereas with hydroxylamine such an intermediate could not be detected.
We have used representational difference analysis (RDA) for subtractive hybridization of oligo dT primed directionally cloned cDNA libraries from human inner ear tissue and a B-lymphoblast cell line. Two rounds of subtraction-amplification, followed by differential hybridization of selected clones led to the isolation of genes which were specific to the ear. Sequence analysis of randomly chosen clones revealed the presence of a histidine rich Ca2+ binding protein, human dynamin, collagen type 1A1, collagen type 2A1, SPARC, human growth hormone, and several specific genes which had no sequence homology in the data base. Furthermore, to apply these techniques for isolating genes specific to distinct inner ear structures and/or cell types of inner ear for which the starting tissue material is limiting, we have used a modified PCR based protocol to construct representative cDNA libraries. We have characterized a cDNA library constructed from small amounts of inner ear tissues recovered by ablative surgical procedure involving labyrinthectomy. The potential application of these protocols for isolating genes involved in hearing and deafness is discussed.
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