Satyam Nampalli

DNA Polymerase Incorporation of Universal Base Triphosphates

Abstract The 5′-triphosphates of various universal deoxynucleosides have been synthesised, and their ability to act as template directed substrates for various polymerases has been investigated. This paper is dedicated with great regard to the late Professor Tsujiaki Hata

TFA-NHS as bifunctional protecting agent: simultaneous protection and activation of amino carboxylic acids

Reaction of amino carboxylic acids with N-trifluoroacetoxy succinimide yields the corresponding trifluoroacetyl protected active NHS esters.

Fluorescence resonance energy transfer terminators for DNA sequencing

A four-colour set of fluorescence resonance energy transfer dideoxy nucleotide terminators (10–13), have been synthesised using a rigid and linear tri-functional molecule (2), synthesised via Heck coupling reaction of t-Boc-l-4-iodophenylalanine (1) with N-TFA-propargylamine. Evaluation of the terminators in DNA sequencing reactions, in combination with Thermo Sequenase™ II DNA polymerase, demonstrated them to be excellent reagents for high-throughput DNA sequencing.

Unnatural amino acid derived FRET cassettes, terminators and their DNA sequencing potential

Abstract An unnatural, tri-functional amino acid , t -Boc- l - para -amino-phenylalanine ( 1 ), was utilized in the design and syntheses of highly efficient FRET cassettes ( 4 – 7 ) and converted them into 2′,3′-dideoxynucleotide terminators ( 8 – 11 ) for investigating DNA sequencing potential coupled with a thermostable DNA polymerase, Thermo Sequenase™ II.

FOUR COLOR FRET DYE NUCLEOTIDE TERMINATORS FOR DNA SEQUENCING

The synthesis of four color set of energy transfer-dye terminators (8a–8d) starting from p-iodo-β-phenylalanine was accomplished and their utility in the sequencing reactions has been evaluated.

Efficient synthesis of 8-oxo-dGTP: a mutagenic nucleotide.

An efficient synthesis of mutagenic and oxidative DNA damage product, 8-oxo-dGTP (4) has been achieved in high yield, along with a serendipitous generation of 8-dimsyl-dG (2). In combination with dPTP (5), 8-oxo-dGTP (4) can be formulated into a kit for investigating DNA random mutagenesis.

Synthesis and enzymatic incorporation of a novel, bicyclic pyrimidine nucleoside: a thymidine mimic

Abstract Nucleophilic ring-opening and rearrangement reaction of a furanopyrimidine nucleoside with anhydrous hydrazine provided a novel, 6,6-bicyclic pyrimidopyridazin-7-one nucleoside (dH, 4 ), whose structure was confirmed by X-ray crystallography. This novel nucleoside was converted to its 5′-triphosphate (dHTP) for studies with DNA polymerases and incorporated into a template by using standard phosphoramidite chemistry. In the template, dH directed the incorporation of dATP and to a lesser extent dGTP into the transcript and dHTP was efficiently incorporated at the 3′-end of a primer opposite dA using both exonuclease free Klenow fragment (KF exo-) and Taq DNA polymerases and extended with natural dNTPs.

Synthesis and incorporation of pyrrole carboxamide nucleoside triphosphates by DNA polymerases.

Abstract We have synthesised and examined the enzymatic incorporation properties of the 5′-triphosphates of 2′-deoxyribosyl pyrrole 3-monocarboxamide (dMTP) and 2′-deoxyribosyl pyrrole 3,4-dicarboxamide (dDTP). These analogues we had hoped would behave as ambivalent base analogues in that they can present two alternative hydrogen-bonding faces either by rotation about the carboxamide group or about the glycosidic bond. The two pyrrole derivatives, dMTP and dDTP, exhibit a preference for incorporation with Klenow polymerase. They are preferentially incorporated as either A or C.

FLUORESCENCE RESONANCE ENERGY TRANSFER DYE NUCLEOTIDE TERMINATORS: A NEW SYNTHETIC APPROACH FOR HIGH-THROUGHOUT DNA SEQUENCING

Fluorescence resonance energy transfer (FRET) based dye-nucleotide terminators (10–13) were designed, synthesized, and formulated with Thermo Sequenase™ II DNA polymerase into a robust kit for high throughput DNA sequencing. The key energy transfer (ET) rigid and linear linker (2), required for the syntheses of energy transfer cassettes (6–9) was synthesized via Heck coupling reaction on t-Boc-L-4-iodo-phenylalanine (1) with N-TFA-propargylamine.

Synthesis and Energy Transfer Efficiency of FRET Terminators Derived from Different Linkers

Abstract A number of different energy transfer dye labeled-cassettes were synthesized using aminoacid based trifunctional linkers and coupled to the propargylamino-substituted dideoxynucleoside-5′-triphosphates (ddNTPs). These terminators were evaluated for their energy transfer efficiency and DNA sequencing potential using thermostable DNA polymerase.