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Results for “duplex sequencing”

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PubMed1999

Cytosine methylation in a CpG sequence leads to enhanced reactivity with Benzo[a]pyrene diol epoxide that correlates with a conformational change.

Benzo[a]pyrene (B[a]P) is a widespread environmental carcinogen that must be activated by cellular metabolism to a diol epoxide form (BPDE) before it reacts with DNA. It has recently been shown that BPDE preferentially modifies the guanine in methylated 5'-CpG-3' sequences in the human p53 gene, providing one explanation for why these sites are mutational hot spots. Using purified duplex oligonucleotides containing identical methylated and unmethylated CpG sequences, we show here that BPDE preferentially modified the guanine in hemimethylated or fully methylated CpG sequences, producing between 3- and 8-fold more modification at this site. Analysis of this reaction using shorter duplex oligonucleotides indicated that it was the level of the (+)-trans isomer that was specifically increased. To determine if there were conformational differences between the methylated and unmethylated B[a]P-modified DNA sequences that may be responsible for this enhanced reactivity, a native polyacrylamide gel electrophoresis analysis was carried out using DNA containing isomerically pure B[a]P-DNA adducts. These experiments showed that each adduct resulted in an altered gel mobility in duplex DNA but that only the presence of a (+)-trans isomer and a methylated C 5' to the adduct resulted in a significant gel mobility shift compared with the unmethylated case.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed2004

Selective formation of stable triplexes including a TA or a CG interrupting site with new bicyclic nucleoside analogues (WNA).

Triplex-forming oligonucleotides (TFOs) are potential DNA-targeting molecules and would become powerful tools for genomic research. As the stabilization of the TFO is partially provided by hydrogen bonds to purine bases, the most stable triplexes form with homopurine/homopyrimidine sequences, and a pyrimidine base in the purine strand of the duplex interrupts triplex formation. If a TFO can recognize sequences including such an interrupting site, the target regions in the genome would be expanded to a greater extent. However, this problem has not been generally solved despite extensive studies. We have previously reported a new base analogue (WNA) constructed of three parts, a benzene ring, a heterocyclic ring, and a bicyclic skeleton to hold these two parts. In this study, we have further investigated modification of WNA systematically and determined two useful WNA analogues, WNA-beta T and WNA-beta C, for selective stabilization of triplexes at a TA and a CG interrupting site, respectively. The triplexes with WNA analogues have exhibited an interesting property in that they are more stable than natural-type triplexes even at low Mg(2+) concentration. From comparison of the results with H-WNA-beta T lacking benzene and those with WNA-H without thymine, it has been suggested that benzene is a major contributor for triplex stability and thymine provides selectivity. Thus, it has been successfully demonstrated that WNA-beta T/TA and WNA-beta C/CG combinations may expand triplex recognition codes in addition to the natural A/AT and G/GC base triplet codes. The results of this study will provide useful information for the design of new WNA analogues to overcome inherent problems for further expansion of triplex recognition codes.

Base Pairing↗

PubMed1993

The 5'-terminal region of the apocytochrome b transcript in Crithidia fasciculata is successively edited by two guide RNAs in the 3' to 5' direction.

We analyzed the chimeric guide RNA (gRNA)-mRNA molecules in Crithidia fasciculata that are predicted to transiently exist in editing of the 5'-terminal domain of apocytochrome b (CYb) mRNA, by polymerase chain reaction amplification and DNA sequencing, and obtained evidence suggesting that among the 14 editing sites numbered from 3' to 5', the sequence in the 3'-half of the sites (3' block) was specified by one guide RNA species (gRNA-I) and that in the remaining half of the sites (5' block) by the other guide RNA species (gRNA-II) and that the direction of editing in each block was 3' to 5'. The predicted transition site of editing by two gRNAs was between the first and second U residues from the 3' end within editing site 7. We found that a stretch of the edited sequence in the 3' block of mRNA could form a stable duplex with a stretch immediately upstream of the guide sequence in gRNA-II. The result leads to a successive editing model that the 3' block of pre-edited mRNA is first edited by gRNA-I, and after completion of editing, the 5' portion of gRNA-II pairs with the edited mRNA for editing of the 5' block.

Animals↗

PubMed1997

Quantitating tertiary binding energies of 2' OH groups on the P1 duplex of the Tetrahymena ribozyme: intrinsic binding energy in an RNA enzyme.

Binding of the Tetrahymena ribozyme's oligonucleotide substrate (S) involves P1 duplex formation with the ribozyme's internal guide sequence (IGS) to give an open complex, followed by docking of the P1 duplex into the catalytic core via tertiary interactions to give a closed complex. The overall binding energies provided by 2' OH groups on S and IGS have been measured previously. To obtain the energetic contribution of each of these 2' OH groups in the docking step, we have separately measured their contribution to the stability of a model P1 duplex using "substrate inhibition". This new approach allows measurement of duplex stabilities under conditions identical to those used for ribozyme binding measurements. The tertiary binding energies from the individual 2' OH groups include a small destabilizing contribution of 0.7 kcal/mol and stabilizing contributions of up to -2.9 kcal/mol. The energetic contributions of specific 2' OH groups are discussed in the context of considerable previous work that has characterized the tertiary interactions of the P1 duplex. A "threshold" model for the open and closed complexes is presented that provides a framework to interpret the energetic effects of functional group substitutions on the P1 duplex. The sum of the tertiary stabilization provided by the conserved G x U wobble at the cleavage site and the individual 2' OH groups on the P1 duplex is significantly greater than the observed tertiary stabilization of S (11.0 vs 2.2 kcal/mol). It is suggested that there is an energetic cost for docking the P1 duplex into the active site that is paid for by the "intrinsic binding energy" of groups on the P1 duplex. Substrates that lack sufficient tertiary binding energy to overcome this energetic barrier exhibit reduced reactivities. Thus, the ribozyme appears to use the intrinsic binding energy of groups on the P1 duplex for catalysis. This intrinsic binding energy may be used to position reactants within the active site and to induce electrostatic destabilization of the substrate, relative to its interactions in solution.

Animals↗

PubMed1999

Characterization of a local folding event of the Tetrahymena group I ribozyme: effects of oligonucleotide substrate length, pH, and temperature on the two substrate binding steps.

Binding of the Tetrahymena group I ribozyme's oligonucleotide substrate occurs in two steps: P1 duplex formation with the ribozyme's internal guide sequence which forms an "open complex" is followed by docking of the P1 duplex into tertiary interactions within the catalytic core which forms a "closed complex". By systematically varying substrate length, pH, and temperature, we have identified conditions under which P1 duplex formation, P1 docking, or the chemical cleavage step limits the rate of the ribozyme reaction. This has enabled characterization of the individual steps as a function of substrate length, pH, and temperature, leading to several conclusions. (1) The rate constant for formation of the open complex is largely independent of substrate length, pH, and temperature, analogous to that of duplex formation in solution. This extends previous results suggesting that open complex formation entails mainly secondary structure formation and strengthens the view that the second binding step, P1 docking, represents a simple transition from secondary to tertiary structure in the context of an otherwise folded RNA. (2) The temperature dependence of the rate constant for P1 docking yields a negative activation entropy, in contrast to the positive entropy change previously observed for the docking equilibrium. This suggests a model in which tertiary interactions are not substantially formed in the transition state for P1 docking. (3) Shortening the substrate by three residues decreases the equilibrium constant for P1 docking by 200-fold, suggesting that the rigidity imposed by full-length duplex formation facilitates formation of tertiary interactions. (4) Once docked, shortened substrates are cleaved at rates within 3-fold of that for the full-length substrate. Thus, all the active site interactions required to accelerate the chemical cleavage event are maintained with shorter substrates. (5) The rate constant of approximately 10(3) min(-1) obtained for P1 docking is significantly faster than the other steps previously identified in the tertiary folding of this RNA. Nevertheless, P1 docking presumably follows other tertiary folding steps because the P1 duplex docks into a core that is formed only upon folding of the rest of the ribozyme.

Animals↗

PubMed2005

Rapid and cost-effective antenatal diagnosis of haemoglobin Bart's hydrops foetalis syndrome using a duplex-polymerase chain reaction.

Haemoglobin Bart's hydrops foetalis syndrome (--SEA/--SEA) is not compatible with life and contributes to a majority of the hydropic foetuses in the Malaysian Chinese alpha-thalassaemia carriers who possess the 2-alpha-gene deletion in cis (--SEA/alphaalpha). A duplex-PCR which simultaneously amplifies a normal 136 bp sequence between the psialpha-alpha2-globin genes and a 730 bp Southeast Asian deletion-specific sequence (--SEA) between the psialpha2-theta1-globin genes was established. The duplex-PCR which detects the --SEA deletion in both chromosomes serves as a rapid and cost-effective confirmatory test in the antenatal diagnosis of Haemoglobin Bart's hydrops foetalis syndrome in Malaysia. In addition, the duplex-PCR is simple to perform as both the normal and deletion-specific alpha-globin gene sequences are amplified in the same PCR reaction.

Chorionic Villi Sampling↗

PubMed1999

Interlocked mismatch-aligned arrowhead DNA motifs.

BACKGROUND: Triplet repeat sequences are of considerable biological importance as the expansion of such tandem arrays can lead to the onset of a range of human diseases. Such sequences can self-pair via mismatch alignments to form higher order structures that have the potential to cause replication blocks, followed by strand slippage and sequence expansion. The all-purine d(GGA)n triplet repeat sequence is of particular interest because purines can align via G.G, A.A and G.A mismatch formation. RESULTS: We have solved the structure of the uniformly 13C,15N-labeled d(G1-G2-A3-G4-G5-A6-T7) sequence in 10 mM Na+ solution. This sequence adopts a novel twofold-symmetric duplex fold where interlocked V-shaped arrowhead motifs are aligned solely via interstrand G1.G4, G2.G5 and A3.A6 mismatch formation. The tip of the arrowhead motif is centered about the p-A3-p step, and symmetry-related local parallel-stranded duplex domains are formed by the G1-G2-A3 and G4-G5-A6 segments of partner strands. CONCLUSIONS: The purine-rich (GGA)n triplet repeat sequence is dispersed throughout the eukaryotic genome. Several features of the arrowhead duplex motif for the (GGA)2 triplet repeat provide a unique scaffold for molecular recognition. These include the large localized bend in the sugar-phosphate backbones, the segmental parallel-stranded alignment of strands and the exposure of the Watson-Crick edges of several mismatched bases.

Base Pair Mismatch↗

PubMed2002

DNA sequence recognition by the indolocarbazole antitumor antibiotic AT2433-B1 and its diastereoisomer.

The antibiotic AT2433-B1 belongs to a therapeutically important class of antitumor agents. This natural product contains an indolocarbazole aglycone connected to a unique disaccharide consisting of a methoxyglucose and an amino sugar subunit, 2,4-dideoxy-4-methylamino-L-xylose. The configuration of the amino sugar distinguishes AT2433-B1 from its diastereoisomer iso-AT2433-B1. Here we have investigated the interaction of these two disaccharide indolocarbazole derivatives with different DNA sequences by means of DNase I footprinting and surface plasmon resonance (SPR). Accurate binding measurements performed at 4 and 25 degrees C using the BIAcore SPR method revealed that AT2433-B1 binds considerably more tightly to a hairpin oligomer containing a [CG](4) block than to an oligomer with a central [AT](4) tract. The kinetic analysis shows that the antibiotic dissociates much more slowly from the GC sequence compared to the AT one. Preferential binding of AT2433-B1 to GC-rich sequences in DNA was independently confirmed by DNase I footprinting experiments performed with a 117 bp DNA restriction fragment. The specific binding sequence 5'-AACGCCAG identified from the footprints was then converted into a biotin-labeled DNA hairpin duplex and compound interactions with this specific sequence were characterized by high resolution BIAcore SPR experiments. Such a combined approach provided a detailed understanding of the molecular basis of DNA recognition. The discovery that the glycosyl antibiotic AT2433-B1 preferentially recognizes defined sequences offers novel opportunities for the future design of sequence-specific DNA-reading small molecules.

Aminoglycosides↗

PubMed1999

A model of EcoRII restriction endonuclease action: the active complex is most likely formed by one protein subunit and one DNA recognition site.

To elucidate the mechanism of interaction of restriction endonuclease EcoRII with DNA, we studied by native gel electrophoresis the binding of this endonuclease to a set of synthetic DNA-duplexes containing the modified or canonical recognition sequence 5'-d(CCA/TGG)-3'. All binding substrate or substrate analogues tested could be divided into two major groups: (i) duplexes that, at the interaction with endonuclease EcoRII, form two types of stable complexes on native gel in the absence of Mg2+ cofactor; (ii) duplexes that form only one type of complex, observed both in the presence and absence of Mg2+. Unlike the latter, duplexes under the first group can be hydrolyzed by endonuclease. Data obtained suggest that the active complex is most likely formed by one protein subunit and one DNA recognition sequence. A model of EcoRII endonuclease action is presented.

Base Sequence↗

PubMed2006

The SL1-SL2 (stem-loop) domain is the primary determinant for stability of the gamma retroviral genomic RNA dimer.

Retroviral genomes are assembled from two sense-strand RNAs by noncovalent interactions at their 5' ends, forming a dimer. The RNA dimerization domain is a potential target for antiretroviral therapy and represents a compelling RNA folding problem. The fundamental dimerization unit for the Moloney murine sarcoma gamma retrovirus spans a 170-nucleotide minimal dimerization active sequence. In the dimer, two self-complementary sequences, PAL1 and PAL2, form intermolecular duplexes, and an SL1-SL2 (stem-loop) domain forms loop-loop base pairs, mediated by GACG tetraloops, and extensive tertiary interactions. To develop a framework for assembly of the retroviral RNA dimer, we quantified the stability of and established nucleotide resolution secondary structure models for sequence variants in which each motif was compromised. Base pairing and tertiary interactions between SL1-SL2 domains contribute a large free energy increment of -10 kcal/mol. In contrast, even though the PAL1 and PAL2 intermolecular duplexes span 10 and 16 bp in the dimer, respectively, they contribute only -2.5 kcal/mol to stability, roughly equal to a single new base pair. First, these results emphasize that the energetic costs for disrupting interactions in the monomer state nearly balance the PAL1 and PAL2 base pairing interactions that form in the dimer. Second, intermolecular duplex formation plays a biological role distinct from simply stabilizing the structure of the retroviral genomic RNA dimer.

Base Sequence↗

PubMed1992

Sequence-dependent distortions induced in DNA by monofunctional platinum(II) binding.

The effects on thermal stability and conformation of DNA produced by the monofunctional adducts of chlorodiethylenetriamineplatinum(II) chloride ([Pt(dien)Cl]Cl) have been investigated. Oligodeoxyribonucleotide duplexes of varying lengths (9-20 base pairs) and of varying central trinucleotide sequences were prepared and characterized that contained site-specific and unique N(7)-guanine adducts. Included are adducts at the sequences of d(AGC), d(AGT), d(CGA), d(TGA), d(TGC), and d(TGT). All these monofunctional adducts decrease the melting temperature (Tm) of the duplexes. This destabilization effect exhibits a sequence-dependent variability. The highest lowering of Tm is observed for the modified duplexes containing the central sequence of pyrimidine-guanine-pyrimidine. The destabilization effect is reduced with decreasing concentrations of Na+. Polarography, circular dichroism, phenanthroline-copper, and chemical probes reveal conformational distortions spreading over several base pairs around the adduct. The effects of monofunctional platinum(II) adducts on conformational distortions in DNA exhibit a sequence-dependent variability similar to those on thermal stability of DNA. The influence of the monofunctional adduct formed by cis-diamminemonoaquamonochloroplatinum(II) on the stability of the oligonucleotide duplex has been also studied. This lesion decreases thermal stability of DNA in the same way as does the adduct of [Pt(dien)Cl]Cl.

Base Sequence↗

PubMed2006

Influence of heterozygosity for parkin mutation on onset age in familial Parkinson disease: the GenePD study.

BACKGROUND: The PARK2 gene at 6q26 encodes parkin, whose inactivation is implicated in an early-onset autosomal recessive form of Parkinson disease (PD). OBJECTIVE: To evaluate the influence of heterozygosity for parkin mutation on onset age in a sample of families with at least 2 PD-affected members. DESIGN: Clinical and genetic study. SETTING: Twenty collaborative clinical sites. PATIENTS: Patients with familial PD collected in the GenePD study. Studied families were selected for (1) affected sibling pairs sharing 2 alleles identical by state at PARK2 (D6S305) or (2) 1 or more family members with onset age younger than 54 years, regardless of D6S305 status. At least 1 member from each of 183 families underwent comprehensive screening for deletion/insertion variants and point mutations in PARK2. MAIN OUTCOME MEASURES: Mutations in the parkin gene were screened by means of single-stranded conformation polymorphism and sequencing in all 12 coding exons and flanking intronic sequences for point mutations and duplex quantitative polymerase chain reaction in all exons for rearrangement, duplication, and deletion. RESULTS: Mutations were found in 23 families (12.6% of those screened). Among the mutation-positive families, 10 (43%) contained compound heterozygotes; 3 (13%), homozygotes; and 10 (43%), heterozygotes. The onset age in patients with parkin gene mutations ranged from 20 to 76 years. Patients with 1 parkin mutation had an 11.7-year age at onset than did patients with none (P = .04), and patients with 2 or more parkin mutations had a 13.2-year decrease in age at onset compared with patients with 1 mutation (P = .04). CONCLUSIONS: These data indicate that parkin mutations are not rare in multiply affected sibships, and that heterozygous mutation carrier status in PARK2 significantly influences age at onset of PD.

Adult↗

PubMed1999

How Do Charges Travel through DNA?-An Update on a Current Debate.

The effects of oligonucleotide sequence, base pairing, stacking, and duplex microstructure as well as donor-acceptor distance, labeling site, and driving force on charge-transfer reactions in DNA are of interest, yet relatively few experiments have been performed to address many of these issues. This article summarizes recent results on DNA-mediated charge transfer and highlights experimental and theoretical issues that are still unanswered or unresolved.

Journal Article↗

PubMed1995

Helical parameters, fluctuations, alternative hydrogen bonding, and bending in oligonucleotides containing a mistmatched base-pair by NOESY distance restrained and distance free molecular dynamics.

We have analysed and compared the molecular structures and dynamics of the DNA duplex, that corresponds to the sequence 29 to 39 of the K-ras gene, where the central base-pair is the normal C.G base or a mismatch base-pair C.A, C.A+, A.G and A+.G. Molecular dynamics runs of 100 picoseconds without or with distance restraints derived from NOE measurements are analysed and compared in all cases. (1) The EMBO convention of helical parameters for nucleic acids is extended to account for the construction and the description of any DNA mismatched base-pair. (2) Both types of MD runs reproduce very well all NMR data, except the H8 H2 inter-residue distances where agreement is not as good. (3) Average parameter values and fluctuations are in good agreement with results derived from persistence length and torsion modulus measurements. (4) Our molecular dynamics suggest the presence, in certain cases, of three-centred hydrogen bonds, which should be viewed as an equilibrium between hydrogen-bonding alternatives. In the case of the C.A mismatch, we observe the correlation between transient DNA bending and possible hydrogen bonding between a base and its 5' neighbour on the opposite strand in the sequence CCA. (5) These molecular dynamics analyses and observations provide a coherent view for the results obtained from recent DNA crystal structures and for results derived from other techniques such as gel electrophoresis at C.A steps.

Base Sequence↗

PubMed1995

Probing the domain structure of the type IC DNA methyltransferase M.EcoR124I by limited proteolysis.

Limited proteolysis has been used to probe the domain structure of the type I DNA methyltransferase M.EcoR124I. Trypsin digestion of the methyltransferase generates two fragments derived from the HsdS subunit, a 28 kDa N-terminal domain and a 19 kDa C-terminal domain, leaving the HsdM subunit intact. Extensive digestion by chymotrypsin, however, removes 59 amino acid residues from the N terminus of the HsdM subunit to leave a 52 kDa C-terminal domain. Binding of the cofactor S-adenosyl methionine has no appreciable effect on the rate of cleavage, but binding of a 30 bp DNA duplex containing the cognate recognition sequence confers almost total protection. Following trypsin cleavage of the methyltransferase, a stable proteolytic product is produced which has been purified for biochemical characterisation. The trypsinised enzyme is shown to be a multimeric complex containing two intact HsdM subunits and both fragments of the HsdS subunit, consistent with the circular model proposed for the organisation of domains in the specificity subunit in type IC methyltransferases. Gel retardation studies show that the proteolysed enzyme still retains DNA binding activity, but its specificity for the DNA recognition sequence is dramatically reduced.

Amino Acid Sequence↗

PubMed1999

Conformational changes of the BS2 operator DNA upon complex formation with the Antennapedia homeodomain studied by NMR with 13C/15N-labeled DNA.

The NMR structures have been determined for a 13C/15N doubly labeled 14 base-pair DNA duplex comprising the BS2 operator sequence both free in solution and in the complex with the Antennapedia homeodomain. The impact of the DNA labeling is assessed from comparison with a previous structure of the same complex that was determined using isotope labeling only for the protein. Differences between the two structure determinations are nearly completely limited to the DNA, which retains the global B -conformation of the free DNA also in the complex. Local protein-induced conformational changes are a narrowing of the minor groove due to the interaction with the N-terminal arm of the homeodomain, and changes of the sugar puckers of the deoxyriboses G5 and C6, which are apparently induced by van der Waals interactions with Tyr25, and with Gln50 and Arg53, respectively. The high conservation of these amino acid residues in homeodomains suggests that protein-induced shifts in some sugar puckers contribute to the affinity of homeodomains to their cognate DNA. The data obtained here with the Antennapedia homeodomain-DNA complex clearly show that nucleic acid isotope-labeling can support detailed conformational characterization of DNA in complexes with proteins, which will be indispensable for structure determinations of complexes containing globally distorted DNA conformations.

Antennapedia Homeodomain Protein↗

PubMed1999

Melanoplus sanguinipes entomopoxvirus DNA topoisomerase: site-specific DNA transesterification and effects of 5'-bridging phosphorothiolates.

Melanoplus sanguinipes entomopoxvirus (MsEPV) encodes a 328 amino acid polypeptide related to the type I topoisomerases of six other genera of vertebrate and insect poxviruses. The gene encoding MsEPV topoisomerase was expressed in bacteria, and the recombinant protein was purified by ion-exchange chromatography and glycerol gradient sedimentation. MsEPV topoisomerase, a monomeric protein, catalyzed the relaxation of supercoiled plasmid DNA at approximately 0.6 supercoils/s. Like other poxvirus topoisomerases, the MsEPV enzyme formed a covalent adduct with duplex DNA at the target sequence CCCTT downward arrow. The kinetic and equilibrium parameters of the DNA transesterification reaction of MsEPV topoisomerase were k(cl) = 0.3 s(-1) and K(cl) = 0.25. The introduction of a 5'-bridging phosphorothiolate at the scissile phosphate increased the cleavage equilibrium constant from 0.25 to >/=30. Similar phosphorothiolate effects were observed with vaccinia topoisomerase. Kinetic analysis of single-turnover cleavage and religation reactions established that the altered equilibrium was the result of a approximately 10(-4) decrement in the rate of topoisomerase-catalyzed attack of 5'-SH DNA on the DNA-(3'-phosphotyrosyl)-enzyme intermediate. 5'-bridging phosphorothiolates at the scissile phosphate and other positions within the CCCTT element had no significant effect on k(cl).

Amino Acid Sequence↗

PubMed2005

Rapid genotyping for relevant CYP1A2 alleles by pyrosequencing.

OBJECTIVE: To develop a rapid and reliable screening method for identifying the relevant cytochrome P450 (CYP) 1A2 alleles CYP1A2*1D (-2467Tdel), *1F (-163A>C), and *1K (-739T>G, -729C>T, -163A>C) that are in linkage disequilibrium with the functionally relevant CYP1A2 polymorphisms and therefore are considered to be predictive for the CYP1A2 phenotype. METHODS: CYP1A2 single nucleotide polymorphisms (SNPs) -2467Tdel, -739T>G, -729C>T, and -163A>C were screened for in 495 healthy Caucasian volunteers using newly developed pyrosequencing duplex and simplex assays. Conventional sequencing of randomly selected samples served as quality control. RESULTS: Frequencies were 7.9% for CYP1A2*1D, 31.8% for *1F, and 0.4% for *1K. The observed distribution of homozygous and heterozygous carriers of the alleles corresponded to the predicted one according to the Hardy-Weinberg law. It also corresponded to reported allelic frequencies from Caucasians but differed significantly from the distribution seen in other ethnicities. The most frequent haplotype was -2467T/-739T/-729C/-163A (allelic frequency 61.6%), followed by -2467T/-739T/-729C/-163C (30.5%), -2467Tdel/-739T/-729C/-163A (5.1%), -2467Tdel/-739G/-729C/-163A (1.2%), and -2467Tdel/-739T/-729C/-163C (1.1%). Complete linkage disequilibrium (value of D' nearly 1) existed between -2467Tdel, -739T>G, and -729C>T and between -729T>G and -163A>C. CONCLUSIONS: Pyrosequencing facilitates rapid and reliable detection of those CYP1A2 alleles that, based on current knowledge, can be considered predictive for the CYP1A2 phenotype.

Alleles↗