Termini Of DNA Fragments Research Articles

New Triphosphate Conjugates Bearing Reporter Groups: Labeling of DNA Fragments for Microarray Analysis

A simple and convenient method for incorporation of fluorescent or ligand groups into 3'-termini of DNA fragments is proposed. A set of triphosphoric acid monoesters bearing fluorescent groups or biotin attached to the triphosphate fragment through linkers of different lengths and structures was synthesized. All the compounds were substrates for calf thymus terminal deoxynucleotidyltransferase and were used for incorporation of marker groups into 3'-termini of DNA fragments. The compounds were successfully applied for DNA labeling during post-PCR target preparation for microarray analysis.

DNA fingerprinting involving fluorescence-labeled termini of any enzymatically generated fragments of DNA

We have developed a new fluorescence-based method for DNA fingerprinting that does not require a fluorescent linker or a synthetic oligonucleotide primer, both of which are normally used for labeling of DNA. Cosmid DNAs are digested with appropriate restriction enzymes and the 3' termini of DNA fragments are labeled with the corresponding, fluorescent dye-conjugated dideoxynucleotide triphosphate terminator (dye-ddNTP) by the Klenow fragment of DNA polymerase I from Escherichia coli, which has 3'-->5' exonuclease and replacement activities as well as its main 5'-->3' polymerase activity. Samples are separated on a DNA-sequencing gel and data are analyzed by application of both the Version 0.3.8a mapper program (Applied Biosystem Inc., Foster City, CA) and our Overlap I program that facilitate rapid analysis of the frequency of overlapping of cosmid DNAs. Using this method we have determined the overlap frequency of DNA fragments of each cosmid clone from the mouse MHC class I gene cluster.

RNA‐Dependent DNA Polymerases

Reverse transcriptases are derived from retroviruses which use them to make DNA copies of their RNA genomes. They are multifunctional enzymes, but they are mainly used as RNA-directed DNA polymerases. Specifically, deoxyoligonucleotides (either oligo(dT) polymers, a collection of random DNA sequences, or a specific sequence) are used as primers for extension on RNA (usually messenger RNA) templates. The DNA synthesized from the RNA template is referred to as complementary DNA (cDNA). This unit describes appropriate reaction conditions along with applications such as synthesizing cDNA for insertion into bacterial cloning vectors, filling in and labeling the 3'termini of DNA fragments with 5' protruding ends, and its use for DNA sequencing in place of Klenow fragment.

Template‐Independent DNA Polymerases

AbstractTerminal deoxynucleotidyl transferase, purified from calf thymus, catalyzes the incorporation of deoxynucleotides to the 3′‐hydroxyl termini of DNA accompanied by the release of inorganic phosphate. Reaction conditions are described in this unit in addition to some applications, including cloning DNA fragments, labeling the 3′ termini of DNA with 32P, incorporating nonradioactive tags onto the 3′ termini of DNA fragments, and synthesizing model polydeoxynucleotide homopolymers.