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Topic: Dideoxynucleotide

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		Dideoxynucleotide use in chain termination sequencing reactions: in a growing DNA chain, DNA polymerase normally adds deoxynucleotides to form a new DNA strand.
		Dideoxynucleotides can also be added, but cause the DNA polymerase to stop DNA synthesis, and thus terminate the growing sequence.
		The sequence can then be read directly from the gel from 5’ to 3’ by looking at the dideoxynucleotide tag from the bottom of the gel to the top (shortest sequence to longest sequence, dideoxynucleotide base by dideoxynucleotide base).
	www.u.arizona.edu /~ajgulbis/MedLinks/Genetics/GeneticsType/17.doc (364 words)

	DNA Sequencing
		Dideoxynucleotides are human-made nucleotides whose sugar component is slightly different from that of the nucleotides that make up DNA.
		As each DNA fragment reaches the bottom of the capillary tube, its fluorescence-labeled end dideoxynucleotide is excited by a laser beam that is directed at the bottom of the tube.
		The 5' terminal base (the dideoxynucleotide) of the shortest fragment (that moves the fastest) is the first base in the electropherogram.
	www.contexo.info /DNA_Basics/dna_sequencing.htm (664 words)

	human genome \| sequencing the DNA
		The addition of a dideoxynucleotide base at the end of a chain stops the addition of any more bases to that end of the chain.
		Each time a dideoxynucleotide base is incorporated, the chain is terminated – no more bases can be added.
		As a result many DNA fragments are made, all of different lengths as the chain terminating bases are added at random during the process.
	www.abpischools.org.uk /resources/poster-series/genome/dnaseq.asp (306 words)

	DNA Sequencing Simulation
		A dideoxynucleotide cannot bond to other nucleotides because its 3' end is modified.
		A primer template, which is a short known sequence of DNA, is attached to the 3' end of the single strand of DNA.
		The DNA polymerase catalyzes the synthesis of a complementary strand of DNA.
	www.accessexcellence.org /AE/AEPC/WWC/1994/dna_sequencing.html (1143 words)

	Sanger Method
		Sanger’s method, which is also referred to as dideoxy sequencing or chain termination, is based on the use of dideoxynucleotides (ddNTP’s) in addition to the normal nucleotides (NTP’s) found in DNA.
		Dideoxynucleotides are essentially the same as nucleotides except they contain a hydrogen group on the 3’ carbon instead of a hydroxyl group (OH).
		However, on occasion a dideoxynucleotide is incorporated into the chain in place of a normal nucleotide, which results in a chain-terminating event.
	www.bio.davidson.edu /Courses/Molbio/MolStudents/spring2003/Obenrader/sanger_method_page.htm (1091 words)

	MCDB 2150 -- Lecture 17
		The dideoxynucleotide that stopped the reaction serves to identify the first nucleotide in the sequence.
		Thus, the first base added to the newly synthesized strand (corresponding to the shortest fragment on the sequencing gel) is complementary to the extreme 3'-position of the template strand, immediately adjacent to the site that the primer hybridizes to.
		The sequence of the template strand can be read down the gel (from longest to shorter fragments) by substituting the base-pairing complement for each of the bases identified by the dideoxynucleotides that were used to terminate the chain growth.
	www.colorado.edu /MCDB/MCDB2150Fall/notes00/L0017.html (1997 words)

	Sequencing Protocols (Site not responding. Last check: 2007-10-31)
		A laser beam directed at the bottom of the capillary tubes excites the fluorescent-labeled dideoxynucleotide which is positioned at the end of the DNA strand.
		A photodetector measures the light emitted by each dideoxynucleotide as it passes through the laser beam.
		The terminal base (the dideoxynucleotides) of the shortest fragment is the first base in the electropherogram.
	www.bscs.org /onco/sprotocol.htm (318 words)

	Nucleotide sequence determination employing matched dideoxynucleotide terminator concentrations (US5273638) (Site not responding. Last check: 2007-10-31)
		A method of positively deciphering a DNA sequence of a sample by comparing data of two electropherograms obtained from separate electrophoresis of products from two complete sequence reactions of the same sample, whereby dideoxynucleotide terminators of different but matched concentration ratios are employed for the two reactions.
		The sequencing chemistry is chosen to result in terminator-concentration dependent in peak intensity in the electropherograms.
		Tabor, Stanley, et al; "Effect of manganese ions on the incorporation of dideoxynucleotides by bacteriophage T7 DNA polymerase and Escherichia coli DNA polymerase l"; Proc.
	www.delphion.com /details?pn10=US05273638 (772 words)

	DNA Sequencing (Site not responding. Last check: 2007-10-31)
		A dideoxynucleotide (dideoxythymidine triphosphate — ddTTP — is the one shown here) can be added to the growing DNA strand but when it is, chain elongation stops because there is no 3′ -OH for the next nucleotide to be attached to.
		Because all four normal nucleotides are present, chain elongation proceeds normally until, by chance, DNA polymerase inserts a dideoxy nucleotide (shown as colored letters) instead of the normal deoxynucleotide (shown as vertical lines).
		Each of the four dideoxynucleotides fluoresces a different color when illuminated by a laser beam and an automatic scanner provides a printout of the sequence.
	home.comcast.net /~john.kimball1/BiologyPages/D/DNAsequencing.html (490 words)

	Recombinant DNA Practice (Site not responding. Last check: 2007-10-31)
		Going from the left-most lane to the right-most lane, the dideoxynucleotide included in each reaction is ddATP, ddCTP, ddGTP, and ddTTP.
		You have to start reading up from the bottom because those pieces of DNA are the smaller fragments.
		They incorporated the dideoxynucleotide early in the in vitro DNA synthesis.
	www.mtsu.edu /~rseipelt/web212b/solutions/212recomb5.htm (84 words)

	DNA analysis
		This reagent is a modified deoxynucleotide triphosphate, where the 3' hydroxyl group is replaced with a hydrogen atom, creating a dideoxynucleotide.
		When the sequencing reaction proceeds in the presence of a small amount of the dideoxynucleotide, the resulting chain-extension reaction products will consist of a set of molecules, all starting at the primer, all extending in the 3' direction, but terminating wherever the dideoxynucleotide happened to be incorporated, opposite its complementary nucleotide in the template.
		Four such ladders were synthesized by use of the four dideoxynucleotides in separate chain extension reactions.
	faculty.washington.edu /dovichi/UBUBTpage/research/Methods/DNA_analysis.html (641 words)

	Biocompare - Product Literature Center
		Better sequence data was obtained with a template purified with the StrataPrep 96 PCR kit, compared to the same PCR template with another commercially available kit.
		After washing to remove contaminants, PCR products are eluted in a small volume of low-ionic-strength buffer and are ready for many applications such as restriction digestion and automated fluorescent dideoxynucleotide sequencing.
		The purified samples were sequenced with the automated fluorescent dideoxynucleotide method, and the data were compared.
	www.biocompare.com /techart.asp?id=199 (1095 words)

	Images of enzyme suggest way to improve DNA sequencing
		But because a nucleotide-like compound called a dideoxynucleotide triphosphate -- ddC, ddG, ddA or ddT -- also is included, Taq stops copying when it incorporates that compound, like a train that halts on the track when it hits a rock.
		Waksman and colleagues obtained X-ray images of Taq at work after crystallizing it with DNA and each of the four dideoxynucleotide triphosphates in turn.
		But the enzyme without arginine at 660 made copies in which every dideoxynucleotide was detectable, so the sequence had no gaps.
	www.eurekalert.org /pub_releases/1999-08/WUSo-Ioes-170899.php (754 words)

	CSUPERB Microchemical Core Facility Homepage
		The automated sequencing method, as developed by ABI Perkin-Elmer for use on the ABI 377 Sequencer, uses PCR to incorporate dideoxynucleotides which contain fluorescent dyes in a primer extension sequencing reaction.
		Each dideoxynucleotide base contains a different fluorescent dye which emits a characteristic wavelength, thus the identity of the dye corresponds to the final base on that fragment.
		The entire reaction is purified, then run in a single lane on a polyacrylamide gel in an ABI 377 Sequencer, so that the fragments separate according to size.
	www.sci.sdsu.edu /dnacore/support.html (548 words)

	Lab #4 of MOLB4170
		The first is the labeling step and the second is the chain-termination step using dideoxynucleotides.
		In the second step, the concentration of all the deoxynucleoside triphosphates is increased and a dideoxynucleoside triphosphate is added.
		Processive DNA synthesis occurs until all growing chains are terminated by a dideoxynucleotide.
	w3.uwyo.edu /~uwmbio/molb4170/lab4.html (1412 words)

	Abstract 55 - Third HIV DRP Symposium: Antiviral Drug Resistance
		This was performed by comparing the peak heights of two nucleotides at a single locus in a chromatogram.
		Previous studies have compared sequenase fluorescent dye-labeled dideoxynucleotide system (dye-terminator system) with dye-labeled primer system.
		It was concluded that the dye-labeled primer system is more appropriate for the quantitative detection of mixture of HIV-1 drug resistance mutations due to a greater correlation of peak heights with the amount of relative DNA present.
	web.ncifcrf.gov /campus/symposium/abstract55.html (294 words)

	dideoxy Sequencing of DNA
		Therefore, the bottom-most band indicates that its particular dideoxynucleotide was added first to the labeled primer.
		In Figure 2, for example, the band that migrated the farthest was in the ddATP reaction mixture.
		This figure shows the structure of a dideoxynucleotide (notice the H atom attached to the 3' carbon).
	www.bio.davidson.edu /courses/Bio111/seq.html (668 words)

	TAMRA-ddCTP, >95% from PerkinElmer - USA
		NEN brand fluorescent dideoxynucleotide analogs are 3’-end chain terminators used for DNA sequencing, RFLP mapping, DNA fingerprinting, and point mutation screening analysis.
		Labeled DNA patterns may be obtained by either separating labeled fragments using standard polyacrylamide gel electrophoresis techniques or with primer/template coated microarrays using 96-well microplates or glass slides as solid supports.
		These analogs are available with a variety of fluorophores attached to each of the four dideoxynucleotides to permit maximum assay flexibility.
	las.perkinelmer.com /Catalog/ProductInfoPage.htm?ProductID=NEL473001EA (154 words)

	Texas Red®-5-ddATP, from PerkinElmer - USA
		Since dideoxynucleotides are chain terminators, they result in the analog being extended in a base specific manner onto the 3’-end of the DNA chain.
		Dideoxynucleotide analogs may be used in a variety of applications which allow determination of a genetic profile based on single nucleotide polymorphisms (SNP)
		Analogs labeled with a fluorophore are intended to be detected directly by their fluorescence properties.
	las.perkinelmer.com /Catalog/ProductInfoPage.htm?ProductID=NEL411001EA (129 words)

	Sequence Analysis
		Dideoxynucleotides (ddNTPs) are another form of nucleotide that inhibit extension of the primer.
		DNA polymerase incorporates the nucleotides and dideoxynucleotides into the growing DNA chain.
		Products of the reactions are then run on a gel that separates the DNA fragments by size.
	www.uvm.edu /~cgep/Education/Sequence.html (1074 words)

	Simultaneous Genotyping and Species Identification Using Hybridization Pattern Recognition Analysis of Generic ...
		Arrows indicate locations of PCR amplification primers and all of the primers used for dideoxynucleotide sequencing (see Methods for sequence).
		1) was determined by use of conventional dideoxynucleotide methodology.
		Dideoxynucleotide chain termination sequencing of the rpoB and 16S genes (clones and PCR amplicons) was carried out on ABI
	www.genome.org /cgi/content/full/8/5/435 (5777 words)

	Bio572: DNA sequencing
		Klenow fragment) is added to the tubes, the synthetic reaction proceeds until, by chance, a dideoxynucleotide is incorporated instead of a deoxynucleotide.
		This is a "chain termination" event, because there is a 3' H instead of a 3' OH group.
		The fluorescent dyes are conjugated to dideoxynucleotides, so a chain termination event is marked with a unique chemical group.
	www.csun.edu /~hcbio027/biotechnology/lec3/seq/s.htm (1370 words)

	Deoxy- and dideoxynucleotide discrimination and identification of critical 5' nuclease domain residues of the DNA ... (Site not responding. Last check: 2007-10-31)
		Deoxy- and dideoxynucleotide discrimination and identification of critical 5' nuclease domain residues of the DNA polymerase I from Mycobacterium tuberculosis -- Mizrahi and Huberts 24 (24): 4845 -- Nucleic Acids Research
		Deoxy- and dideoxynucleotide discrimination and identification of critical 5' nuclease domain residues of the DNA polymerase I from Mycobacterium tuberculosis
		The lack of effect of the His-tag on the dideoxynucleotide susceptibility of the DNA polymerase activity of the Mtb enzyme was confirmed by analysing the effect of chain terminators on DNA synthesis catalysed by the non-tagged recombinant.
	intl-nar.oxfordjournals.org /cgi/content/full/24/24/4845 (6112 words)

	HIVtools :: Nucleoside Analogs (NRTIs) (Site not responding. Last check: 2007-10-31)
		Initially, the nucleoside analog biotransformation requires enzymes such as thymidine kinase, deoxycytidine kinase, or inosine phosphotransferase to form the monophosphate metabolite.
		Once in the triphosphorylated form, the dideoxynucleotide can compete with naturally occurring nucleotides for DNA incorporation.
		The dideoxynucleotide will bind onto reverse transcriptase and become incorporated into the elongating viral complement DNA (cDNA).
	www.hivtools.com /NRTI.php (2250 words)

	DNA Sequencing
		The most common method to determine the sequence of DNA is to utilize enzymes and special nucleotides called dideoxynucleotides.
		The dideoxynucleotide concentrations are carefully adjusted so that they are incorporated into the growing DNA strand randomly and infrequently.
		If the concentration of the dideoxynucleotide was too low when it was mixed with the other four deoxynucleotides, how would that affect the sequencing results?
	www.mnstate.edu /provost/DNA_Seq_Outreach.html (1090 words)

	Sequenom
		MassEXTEND Mix 19 Di: The hME Extend Mix 19 provides deoxynucleotides dGTP and dTTP and dideoxynucleotide ddATP and ddCTP.
		MassEXTEND Mix 20 Di: The hME Extend Mix 20 provides deoxynucleotides dCTP and dTTP and dideoxynucleotides ddATP and ddGTP.
		MassEXTEND Mix 23 Di: The hME Extend Mix 23 provides deoxynucleotides dATP and dGTP and dideoxynucleotides ddCTP and ddTTP.
	www.sequenom.com /Files/customer_support/online_catalog.html (591 words)

	ScienceDaily: Images Of Enzyme Suggest Way To Improve DNA Sequencing
		Four genetic letters A, T, C and G make up the DNA code, and all of these nucleotides are included in the reaction mixture when Taq is put to work.
		But because a nucleotide-like compound called a dideoxynucleotide triphosphate ddC, ddG, ddA or ddT also is included, Taq stops copying when it incorporates that compound, like a train that halts on the track when it hits a rock.
		When it interacts with ddG, one amino acid building block in its fingers residue 660 binds to the dideoxynucleotide.
	www.sciencedaily.com /releases/1999/08/990819064940.htm (2020 words)

	Assays for Antiretroviral Resistance (Site not responding. Last check: 2007-10-31)
		Dideoxynucleotide sequencing is the standard approach to HIV genotyping; it involves the synthesis of a new DNA chain in the presence of a primer and a carefully created mix of deoxynucleotide triphosphates (dNTPs) and dideoxynucleotide triphosphate chain terminators (ddNTPs).
		In one study, 13 research laboratories were shipped cell pellets from cultured HIV-1 isolates.(35) The sequence concordance among laboratories was 99.7% at all nucleotide positions and 97% at positions associated with zidovudine resistance.
		Comparative performance of high-density oligonucleotide sequencing and dideoxynucleotide sequencing of HIV type 1 pol from clinical samples.
	hivinsite.ucsf.edu /InSite?page=kb-authors&doc=kb-02-02-03 (7457 words)

	Ask A Scientist - Sanger method of sequencing
		The position at which polymerases begin synthesizing DNA both in the test tube and in living cells is determined by the positioning of a short nucleic acid fragment called a primer that is complementary to the template.
		The Sanger method depends on the use of a mixture of a dideoxynucleotides and deoxynucleotides so that you don't stop at the first instance of any particular nucleotide in the extending strand.
		We'd much rather get products that terminate at every cytosine position, which means that sometimes we want to incorporate a normal nucleotide rather than a dideoxynucleotide.
	www.hhmi.org /cgi-bin/askascientist/highlight.pl?kw=&file=answers/molecular/ans_007.html (815 words)

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