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K V Deugau

Publications and source records attributed to K V Deugau.

9 recordsLinked to original sources
PubMed1994

Non-cloning amplification of specific DNA fragments from whole genomic DNA digests using DNA 'indexers'.

A highly systematic, non-cloning method of distinguishing and isolating every fragment in a class-IIS or interrupted palindrome restriction digest has been developed in our laboratory. These enzymes produce informative, non-identical cohesive ends which can be selectively modified by ligation to individual synthetic oligodeoxyribonucleotides with the corresponding complementary ends. In this way, polymerase chain reaction and sequencing primer sites and labels can be introduced specifically into a single fragment in a total genomic digest. Known and unknown fragments from genomes of the complexity of Escherichia coli can be isolated directly in sequencable form without the necessity of synthesizing unique primers. Human DNA has also been assessed in this way. Problems intrinsic to cloning (selective fragment loss, mutation and sequence rearrangement) are avoided. Systematic characterization of DNA fragments by their cohesive ends and length provides tremendous power and flexibility for analysis of any DNA molecule without specific clones, probes or libraries. We report proof of principle of this remarkable system and indicate potential applications in DNA sequence tagged site and restriction mapping, sequencing, restriction-fragment-length polymorphism analysis and DNA diagnostics.

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PubMed1992

Differential termination of primer extension: a novel, quantifiable method for detection of point mutations.

We have developed a novel technique that is useful for the rapid detection of previously characterized point mutations in the human genome. The method relies on the differential termination of primer extension being performed simultaneously on normal and mutant template molecules in the presence of a selectively limited complement of deoxynucleotide triphosphates. We have used this technique to determine the carrier status of two potential carriers of a hemophilia B mutation at codon 145 in the factor IX gene. In addition, the technique has been used to quantify the levels of mutant sequence in several tissues of a hemophilia B patient who is a somatic mosaic for a point mutation at codon 350.

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PubMed1991

Somatic mosaicism and female-to-female transmission in a kindred with hemophilia B (factor IX deficiency).

Studies have shown that hemophilia B (Christmas disease; factor IX deficiency) results from many different mutations in the factor IX gene, of which greater than 95% are single nucleotide substitutions. This study has identified a previously unreported form of hemophilia B in a patient who was a somatic mosaic for a guanine-to-cytosine transversion at nucleotide 31,170 in the factor IX gene. This point mutation changes the codon for residue 350 in the catalytic domain of factor IX from a cysteine to a serine. We used differential termination of primer extension to confirm and measure the degree of mosaicism. Our study shows that a varying proportion of cells from hepatic, renal, smooth muscle, and hematopoietic populations possessed normal as well as mutant factor IX sequences. These results indicate that the mutation in this patient occurred either as an uncorrected half-chromatid mutation in the female gamete or as a replication or postreplication error in the initial mitotic divisions of the zygote preceding implantation. In addition, this kindred also contains two females in successive generations who have moderately severe factor IX deficiency. The molecular pathogenesis of this latter phenomenon has been studied and seems to relate to the unaccompanied expression of the mutant factor IX gene consequent upon a second, as yet undefined, genetic event that has prevented inactivation of sequences including the mutant factor IX gene on the X chromosome inherited from the affected male.

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PubMed1985

Apolipoprotein C-II mRNA levels in primate liver. Induction by estrogen in the human hepatocarcinoma cell line, HepG2.

We have demonstrated that physiological concentrations of 17 beta-estradiol increase nuclear estrogen-specific binding sites in the human hepatoma cell line HepG2 7- to 10-fold and the rate of accumulation of secreted apolipoprotein C-II (apo-C-II), 2.5-fold (Tam, S-P., Archer, T. K., and Deeley, R. G. (1985) J. Biol. Chem. 260, 1670-1675). Apo-C-II is the major activator of lipoprotein lipase, an enzyme which plays a key role in lipoprotein catabolism. In order to define more precisely the mechanism by which estrogen influences apo-C-II production, we have synthesized a triacontanucleotide DNA probe that is complementary to apo-C-II mRNA. We have used the probe both in Northern hybridization experiments and in DNA excess titrations to quantify apo-C-II mRNA in hormonally treated HepG2 cells and various primate tissues. These studies revealed that: 1) the concentration of apo-C-II mRNA in HepG2 cells is comparable with that present in human liver; 2) treatment of the cells with low levels of estrogen results in a doubling of the apo-C-II mRNA concentration; 3) the apo-C-II mRNA concentration in monkey liver is 60- to 70-fold greater than in the intestine and 2.5-fold higher than in human liver.

Animals↗
PubMed1983

Nucleotide sequence of polypyrimidines from cloned mouse DNA as determined by base-specific blockage of exonuclease action.

Cloned fragments of mouse DNA have been screened for the presence of long polypyrimidine/polypurine segments. The polypyrimidine portion of one such segment (about 200 nucleotides in length) has been isolated by acidic depurination of the entire cloned fragment and plasmid vector followed by selective precipitation and 5'-32P labeling. This polypyrimidine has been used to demonstrate a new procedure for sequencing. Covalent modification of thymine with a water-soluble carbodiimide, or cytosine with glutaric anhydride, at low levels blocked the action of snake venom exonuclease. After deblocking, separation of the products of digestion by polyacrylamide gel electrophoresis yields a sequence ladder which can be used to determine the position of C and T residues as in other sequencing methods. A sequence of 72 residues adjacent to the 5' end has been established, consisting principally of the repeating tetranucleotide (CCTT)n. A low ratio of endonuclease to exonuclease is essential for application of this method to sequences of this size. Accordingly, a very sensitive modification of a fluorometric endonuclease assay was developed and used to optimize pH and Mg2+ conditions to favor exonuclease activity over the accompanying endonuclease activity. The results clearly indicate that long polypyrimidine tracts can be efficiently prepared and their sequences determined with this method using commercially available exonuclease preparations without additional purification.

Animals↗
PubMed1983

An efficient method for the sequence analysis of oligodeoxyribonucleotides.

Modifications of the chemical method of DNA sequence analysis that permit rapid and reliable sequence determination of single-stranded oligodeoxyribonucleotides as short as 4 nucleotides in length are reported. The principal changes made were increasing the level of chemical modification and optimizing the conditions for recovery of the chemically modified oligodeoxyribonucleotides. This method includes two approaches to the removal of [gamma-32P]ATP from 32P-labeled oligodeoxyribonucleotides and is especially useful in the determination of the sequence of chemically synthesized oligodeoxyribonucleotides, which are generally between 4 and 20 nucleotides in length.

Animals↗
PubMed1978

T4 polynucleotide ligase catalyzed joining of short synthetic DNA duplexes at base-paired ends.

The self-complementary octanucleotide dT-A-G-T-A-C-T-A has been synthesized and its sequence confirmed by two-dimensional fingerprinting. Under conditions used for the T4 polynucleotide ligase reaction, this oligonucleotide forms a dimeric duplex which shows a Tm of 18 degrees C. The optimal rate of joining of the 32P-labeled duplex occurs between 12 and 15 degrees C. The rate is highly concentration dependent, as expected for a bimolecular process. Polyacrylamide gel electrophoretic analysis of this reaction shows the presence of products up to 120 nucleotides in length. In a denaturing gel, each product appears as a double band due to the presence of its 5'-adenylylated activated intermediate. Substrates larger than eight base pairs are utilized more rapidly than the eight base pair duplex, indicating that the T4 ligase has a higher affinity for longer substrates. The low level of nicked intermediates suggests that the joining of both strands requires two steps, the rates of which must be similar.

Base Sequence↗
PubMedDate not supplied

A simplified procedure for cDNA and genomic library construction using nonpalindromic oligonucleotide adaptors.

The properties and characteristics of oligonucleotide adaptors for use in a simplified procedure for the construction of cDNA and genomic DNA libraries are described. The adaptors are suitable for joining to blunt ended cDNA or sheared genomic DNA, and then to the cohesive ends of restriction sites in vectors. Each adaptor consists of two oligonucleotides with complementary but nonpalindromic sequences that include an internal restriction site, a 5' phosphorylated blunt end, and an overlapping or staggered 5' hydroxylated end corresponding to a restriction endonuclease site in a vector of choice. Ligation of the blunt end to high molecular weight target DNA proceeds efficiently and there is no tandem concatenation of the adaptor. Insertion into the appropriate vector only requires ligation of the cohesive ends. There is no requirement for methylation, restriction enzyme cleavage, G-C tailing, or denaturation after ligation of the adaptor to the target DNA, all characteristics of other procedures.

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