A method is presented for choosing optimal oligodeoxyribonucleotides as probes for filter hybridization, primers for sequencing, or primers for DNA amplification. Three main factors that determine the quality of a probe are considered: stability of the duplex formed between the probe and target nucleic acid, specificity of the probe for the intended target sequence, and self-complementarity. DNA duplex stability calculations are based on the nearest-neighbor thermodynamic values determined by Breslauer et al. [Proc. Natl. Acad. Sci. U.S.A. (1986), 83: 3746]. Temperatures of duplex dissociation predicted by the method described here were within 0.4 degrees C of the values obtained experimentally for ten oligonucleotides. Calculations for specificity of the probe and its self-complementarity are based on a simple dynamic algorithm.

OLIGO performs a range of functions for researches in PCR and related technologies such as PCR and sequencing primer selection, hybridization probe design, inverse and real-time PCR, analysis of false priming using a unique priming efficiency (PE) algorithm, design of consensus and multiplex, nested primers and degenerate primers, reverse translation, and restriction enzyme analysis and prediction; based on a protein sequence, oligonucleotide database allows fully automatic multiplexing, primer secondary structure analysis, and more. OLIGO allows for sequence file batch processing that is essential for automation. This chapter describes the major functions of OLIGO version 7 software.

In the polymerase chain reaction (PCR) technique, DNA is amplified in vitro by a series of polymerization cycles consisting of three temperature-dependent steps: DNA denaturation, primer-template annealing, and DNA synthesis by a thermostable DNA polymerase. The purity and yield of the reaction products depend on several parameters, one of which is the annealing temperature (Ta). At both sub- and super-optimal Ta values, non-specific products may be formed, and the yield of products is reduced. Optimizing the Ta is especially critical when long products are synthesized or when total genomic DNA is the substrate for PCR. In this article we experimentally determine the optimal annealing temperature (TaOPT) values for several primer-template pairs and develop a method for its calculation. The TaOPT is found to be a function of the melting temperatures of the less stable primer-template pair and of the product. The fact that experimental and calculated TaOPT values agree to within 0.7 degree C eliminates the need for determining TaOPT experimentally. Synthesis of DNA fragments shorter than 1 kb is more efficient if a variable Ta is used, such that the Ta is higher in each consecutive cycle.

One of the most important factors affecting the quality of PCR is the choice of primers. In general, the longer the PCR product the more difficult it is to select efficient primers and set appropriate designing primers, and in general, the more DNA sequence information is available, the better the chance of finding an optimal primer pair. Efficient primers can be designed by avoiding the following flaws: primer-dimer formation, self-complementarity, too low Tm of the primers, and/or their incorrect internal stability profile. Tips on subcloning PCR products, calculating duplex stability (predicting dimer formation strength), and designing degenerate primers are given.

The 25-kDa mRNA cap-binding protein (ClP) involved in translation was purified by afflinity chromatography from human erythrocytes and rabbit reticulocytes. The sequences of eight human and seven rabbit tryptic and V8 proteolytic peptides were determined. Based on the peptide sequence data, oligodeoxynucleotide probes were synthesized and used to screen human fibroblast and lymphocyte X cDNA libraries. The DNA sequence obtained from recombinant X phage inserts was found to code for all but one peptide. A 23-base oligonucleotide was synthesized based on the DNA sequence and used to prime synthesis of cDNA from human placental mRNA to construct a third library in XgtlO. Screening with a 22-base oligonucleotide, whose sequence was upstream from the 23-base primer, yielded numerous recombinant phages with w250-base inserts. The 1900-base-pair cDNA sequence compiled from all phage inserts appeared to represent the entire primary sequence of CBP (Mr 25,117). Blot analysis of human placental and HeLa mRNA revealed multiple CBP mRNA species ranging from 1925 to 2250 bases. The amino acid sequence of CBP showed homology to the cap-binding PB2 protein of influenza virus.The 25-kDa mRNA cap-binding protein (CBP) recognizes and binds to the 7-methylguanosine-containing mRNA "cap" during an early step in the initiation of protein synthesis (1-4). Valuable information on the highly specific interaction between mRNA caps and CBP as well as the interactions of CBP with eukaryotic initiation factors 4A, 4B, and P220 (5-8) could potentially be derived from the primary structure of CBP. A potential regulatory role for CBP, which may act at the rate-limiting step for initiation (9)(10)(11)(12), could also be investigated using cDNA probes to follow changes in the cellular level of CBP mRNA. At another level, CBP activity may be modulated by phosphorylation/dephosphorylation, because CBP is isolated as a mixture of phosphorylated and nonphosphorylated forms (13,14) and because the dephosphorylated form has been correlated with a decrease in protein synthesis (15). Sequence information could be used to define and characterize the site of phosphorylation. Finally, determination of the entire coding region of the CBP mRNA could be used to establish unequivocally the size of the initial translation product, in view of reports of CBPs of higher molecular weight (16)(17)(18)(19)(20).In this report we describe the sequence analysis of tryptic peptides of rabbit and human CBP, the construction of oligonucleotide probes, and the selection and sequence analysis of recombinant X phages from human fibroblast, lymphocyte, and placental cDNA libraries that establish the primary structure of CBP. The mRNA coding for CBP was also detected by hybridization to cloned probes. MATERIALS AND METHODSMaterials. Trypsin treated with L-1-tosylamido-2-phenylethyl chloromethyl ketone was purchased from Cooper Biomedical (Malvern, PA). Protease V8 was obtained from Miles. All solvents used for HPLC were obtained from Fisher and were HPLC grade. Enzymes...