Gene analysis techniques最新文献

Yeast artificial chromosome (YAC) cloning has allowed isolation of much longer DNA fragments than was previously possible. While this technology makes feasible the isolation of large regions of complex genomes in overlapping cloned segments, it has also required altered methods for manipulation of cloned DNAs. In particular, the isolation of insert sequences from YAC clones has been especially difficult. Methods for characterization of YAC inserts are described and compared.

Two different lengths of long unique synthetic oligonucleotide probes (37- and 48-mers) specific for human major histocompatibility complex (MHC) class II beta genes were synthesized. These oligonucleotides were utilized to examine factors influencing hybridization specificity. Both probe length and stringency of washing conditions were found to be crucial factors for sequence-specific hybridization.

A novel, convenient, and highly efficient hybrid selection procedure is described. The method utilizes the polymerase chain reaction (PCR) in which one of two primers is biotinylated at the 5′-terminus. The concentration of the biotinylated primer is 100 times that of the other to synthesize biotinylated single-stranded DNA (asymmetric PCR). After hybridization of the biotinylated DNA with mRNA in solution, streptavidin agarose is used to trap the hybrid duplex of mRNA·DNA-biotin onto the solid matrix. The selected mRNA is then eluted from the streptavidin agarose. The quantitative physical recovery of selected mRNA is about 70% with about 33% retention of biological activity.

A modification of the Barnes “kilo-sequencing” method is described. The procedure presented here makes it possible to obtain a series of nested deletions on large DNA fragments in only two days. It applies to double-stranded DNA, and thus can be used with plasmids as well as the M13mp series of bacteriophages. The main improvements are the use of a second restriction enzyme, which makes it possible to begin the deletions at any site on the DNA fragment, and the use of mung bean nuclease for trimming the DNA edges so that any restriction enzyme can be used. This method, using a pUC vector and sequencing on double-stranded DNA, would make it possible to read a DNA nucleotide sequence on both strands starting with only one construction.

A new method for purification of specific DNA sequences using a solid phase technique has been developed based on a fusion between the Escherichia coli lac repressor gene (lacI) and the staphylococcal protein A gene (spa). The fusion protein, expressed in Escherichiacoli, is active both in vivo and in vitro with respect to its three functional activities (DNA binding, IPTG induction, and IgG binding). The recombinant protein can be immobilized in a one-step procedure with high yield and parity using the specific interaction between protein A and the Fc-part of immunoglobulin G. The immobilized repressor can thereafter be used for affinity purification of specific DNA fragments containing the lac operator (lacO) sequence.

An efficient procedure for random chemical mutagenesis was used to create analogs of human interferon (IFN)-α4. Unique restriction enzyme sites were introduced into the human IFN-α4 gene to enable cassetting of the gene for localized random mutagenesis. Single-stranded IFN-α4 DNA was treated with nitrous acid, followed by second-strand synthesis using reverse transcriptase. A 72 base pair cassette spanning the coding region for amino acid residues 120 to 136 (120–136 region) was isolated and cloned into a phagemid vector adjacent to a GC-rich sequence. A DNA segment comprising the IFN-α4 cassette sequence and the GC clamp was excised and electrophoresed on a denaturing gradient gel, which allowed the separation from unmutated DNA of DNA fragments with single base pair changes. DNA fragments with mobility different from that of the unmutated fragment were pooled and cloned into an expression vector. Using this procedure, mutations were found in the DNA of 48% of the clones analyzed. However, mutations at two “hot spots” accounted for 89% of these clones. Four of the IFN-α4 analogs with mutations in the 120–136 region were expressed in vitro. The antiproliferative activities on human Daudi cells of most of the analogs were less than 0.2% of the activity of unmodified IFN-α4, suggesting that the integrity of the carboxy terminus is important for the antiproliferative activity of human IFN-α4.

cDNA clones of differentially expressed mRNAs in a colon carcinoma and a hepatocellular carcinoma have been isolated by subtractive cDNA cloning. The subtracted material is at least 90 × enriched for differentially expressed sequences and can be used for construction of subtractive cDNA libraries and polymerase chain reaction (PCR) amplification to generate differential probes. Commercially available λZAP II is used for construction of primary libraries since single-stranded phage bearing the cloned cDNA can be excised in vivo and because λ libraries are convenient for subsequent screening and manipulations. Rare mRNAs (less than 0.01% abundance), which are differentially expressed, can be isolated utilizing this procedure.