Kirk BW, Feinsod M, Favis R, Kliman RM, Barany F.
Department of Microbiology, Box 62, Hearst Microbiology Research Center,
Joan and Sanford I. Weill Medical College of Cornell
University, Room B-406, 1300 York Avenue, New York, NY 10021, USA.
Successful investigation of common diseases requires advances in our
understanding of the organization of the genome. Linkage
disequilibrium provides a theoretical basis for performing candidate
gene or whole-genome association studies to analyze complex
disease. However, to constructively interrogate SNPs for these studies,
technologies with sufficient throughput and sensitivity are
required. A plethora of suitable and reliable methods have been developed,
each of which has its own unique advantage. The
characteristics of the most promising genotyping and polymorphism scanning
technologies are presented. These technologies are
examined both in the context of complex disease investigation and in
their capacity to face the unique physical and molecular challenges
(allele amplification, loss of heterozygosity and stromal contamination)
of solid tumor research.
PMID: 12140314 [PubMed - indexed for MEDLINE]
Huang J, Lu J, Barany F, Cao W.
Department of Microbiology and Immunology, Hearst Microbiology Research
Center and Strang Cancer Prevention Center, The Joan
and Sanford I. Weill Medical College of Cornell University, 1300 York
Avenue, Box 62, New York, New York 10021, USA.
Endonuclease V nicks damaged DNA at the second phosphodiester bond 3'
to inosine, uracil, mismatched bases, or abasic (AP)
sites. Alanine scanning mutagenesis was performed in nine conserved
positions of Thermotoga maritima endonuclease V to identify
amino acid residues involved in recognition or endonucleolytic cleavage
of these diverse substrates. Alanine substitution at D43, E89,
and D110 either abolishes or substantially reduces inosine cleavage
activity. These three mutants gain binding affinity for binding to
double-stranded or single-stranded inosine substrates in the absence
of a metal ion, suggesting that these residues may be involved in
coordinating catalytic metal ion(s). Y80A, H116A, and, to a lesser
extent, R88A demonstrate reduced affinities for double-stranded
or single-stranded inosine substrates or nicked products. The lack
of tight binding to a nicked inosine product accounts for the
increased rate of turnover of inosine substrate since the product release
is less rate-limiting. Y80A, R88A, and H116A fail to cleave
AP site substrates. Their activities toward uracil substrates are in
the following order: H116A > R88A > Y80A. These residues may
play a role in substrate recognition. K139A maintains wild-type binding
affinity for binding to double-stranded and single-stranded
inosine substrate, but fails to cleave AP site and uracil substrate
efficiently, suggesting that K139 may play a role in facilitating
non-inosine substrate cleavage.
PMID: 12081482 [PubMed - indexed for MEDLINE]
Huang J, Kirk B, Favis R, Soussi T, Paty P, Cao W, Barany F.
Department of Microbiology, Box 62, Hearst Microbiology Research Center,
Strang Cancer Prevention Center, Joan and Sanford I
Weill Medical College of Cornell University, Room B-406, 1300 York
Avenue, New York, NY 10021, USA.
Knowledge of inherited and sporadic mutations in known and candidate
cancer genes may influence clinical decisions. We have
developed a mutation scanning method that combines thermostable EndonucleaseV
(Endo V) and DNA ligase. Variant and wild-type
PCR amplicons are generated using fluorescently labeled primers, and
heteroduplexed. Thermotoga maritima (Tma) EndoV recognizes
and primarily cleaves heteroduplex DNA one base 3' to the mismatch,
as well as nicking matched DNA at low levels. Thermus species
(Tsp.) AK16D DNA ligase reseals the background nicks to create a highly
sensitive and specific assay. The fragment mobility on a
DNA sequencing gel reveals the approximate position of the mutation.
This method identified 31/35 and 8/8 unique point mutations
and insertions/deletions, respectively, in the p53, VHL, K-ras, APC,
BRCA1, and BRCA2 genes. The method has the sensitivity to
detect K-ras mutations diluted 1 : 20 with wild-type DNA, a p53 mutation
in a 1.7 kb amplicon, and unknown p53 mutations in
pooled DNA samples. EndoV/Ligase mutation scanning combined with PCR/LDR/Universal
array proved superior to automated
DNA sequencing for detecting p53 mutations in colon tumors. This technique
is well suited for scanning low-frequency mutations in
pooled samples and for analysing tumor DNA containing a minority of
the unknown mutation.
PMID: 11896624 [PubMed - indexed for MEDLINE]
Huang J, Lu J, Barany F, Cao W.
Department of Microbiology and Immunology, Hearst Microbiology Research
Center and Strang Cancer Prevention Center, The Joan
and Sanford I. Weill Medical College of Cornell University, 1300 York
Avenue, Box 62, New York, New York 10021, USA.
Endonuclease V is a deoxyinosine 3'-endonuclease which initiates removal
of inosine from damaged DNA. A thermostable
endonuclease V from the hyperthermophilic bacterium Thermotoga maritima
has been cloned and expressed in Escherichia coli. The
DNA recognition and reaction mechanisms were probed with both double-stranded
and single-stranded oligonucleotide substrates
which contained inosine, abasic site (AP site), uracil, or mismatches.
Gel mobility shift and kinetic analyses indicate that the enzyme
remains bound to the cleaved inosine product. This slow product release
may be required in vivo to ensure an orderly process of
repairing deaminated DNA. When the enzyme is in excess, the primary
nicked products experience a second nicking event on the
complementary strand, leading to a double-stranded break. Cleavage
at AP sites suggests that the enzyme may use a combination of
base contacts and local distortion for recognition. The weak binding
to uracil sites may preclude the enzyme from playing a significant
role in repair of such sites, which may be occupied by uracil-specific
DNA glycosylases. Analysis of cleavage patterns of all 12 natural
mismatched base pairs suggests that purine bases are preferrentially
cleaved, showing a general hierarchy of A = G > T > C. A model
accounting for the recognition and strand nicking mechanism of endonuclease
V is presented.
PMID: 11467933 [PubMed - indexed for MEDLINE]
J Natl Cancer Inst 2001 Jun 6;93(11):858-65
Dong SM, Traverso G, Johnson C, Geng L, Favis R, Boynton K, Hibi
K, Goodman SN, D'Allessio M, Paty P, Hamilton
SR, Sidransky D, Barany F, Levin B, Shuber A, Kinzler KW, Vogelstein
B, Jen J.
Division of Head and Neck Cancer Research, Department of Otolaryngology-Head
and Neck Surgery, The Johns Hopkins Medical
School, The Johns Hopkins University, Baltimore, MD, USA.
BACKGROUND: Colorectal cancer cells are shed into the stool, providing
a potential means for the early detection of the disease
using noninvasive approaches. Our goal was to develop reliable, specific
molecular genetic tests for the detection of colorectal cancer
in stool samples. METHODS: Stool DNA was isolated from paired stools
and primary tumor samples from 51 colorectal cancer
patients. Three genetic targets-TP53, BAT26, and K-RAS-were used to
detect tumor-associated mutations in the stool prior to or
without regard to the molecular analyses of the paired tumors. TP53
gene mutations were detected with a mismatch-ligation assay that
detects nine common p53 gene mutations. Deletions within the BAT26
locus were detected by a modified solid-phase minisequencing
method. Mutations in codons 12 and 13 of K-RAS were detected with a
digital polymerase chain reaction-based method. RESULTS:
TP53 gene mutations were detected in the tumor DNA of 30 patients,
all of whom had the identical TP53 mutation in their stools.
Tumors from three patients contained a noninherited deletion at the
BAT26 locus, and the same alterations were identified in these
patients' stool specimens. Nineteen of 50 tumors tested had a K-RAS
mutation; identical mutations were detected in the paired stool
DNA samples from eight patients. In no case was a mutation found in
stool that was not also present in the primary tumor. Thus, the
three genetic markers together detected 36 (71%) of 51 patients (95%
confidence interval [CI] = 56% to 83%) with colorectal cancer
and 36 (92%) of 39 patients (95% CI = 79% to 98%) whose tumors had
an alteration. CONCLUSION: We were able to detect the
majority of colorectal cancers by analyzing stool DNA for just three
genetic markers. Additional work is needed to determine the
specificity of these genetic tests for detecting colorectal neoplasia
in asymptomatic patients and to more precisely estimate the
prevalence of the mutations and sensitivity of the assay.
PMID: 11390535 [PubMed - indexed for MEDLINE]
Nat Biotechnol 2000 May;18(5):561-4
Array-based mutation detection methodology typically relies on direct hybridization of the fluorescently labeled query sequence to surface-bound oligonucleotide probes. These probes contain either small sequence variations or perfect-match sequence. The intensity of fluorescence bound to each oligonucleotide probe is intended to reveal which sequence is perfectly complementary to the query sequence. However, these approaches have not always been successful, especially for detection of small frameshift mutations. Here we describe a multiplex assay to detect small insertions and deletions by using a modified PCR to evenly amplify each amplicon (PCR/PCR), followed by ligase detection reaction (LDR). Mutations were identified by screening reaction products with a universal DNA microarray, which uncouples mutation detection from array hybridization and provides for high sensitivity. Using the three BRCA1 and BRCA2 founder mutations in the Ashkenazi Jewish population (BRCA1 185delAG; BRCA1 5382insC; BRCA2 6174delT) as a model system, the assay readily detected these mutations in multiplexed reactions. Our results demonstrate that universal microarray analysis of PCR/PCR/LDR products permits rapid identification of small insertion and deletion mutations in the context of both clinical diagnosis and population studies.
PMID: 10802632, UI: 20264187
Nucleic Acids Res 2000 Mar 15;28(6):1447-54
An NAD(+)-dependent DNA ligase from the hyperthermophilic bacterium Aquifex aeolicus was cloned, expressed in Escherichia coli and purified to homogeneity. The enzyme is most active in slightly alkaline pH conditions with either Mg(2+)or Mn(2+)as the metal cofactor. Ca(2+)and Ni(2+)mainly support formation of DNA-adenylate intermediates. The catalytic cycle is characterized by a low k(cat)value of 2 min(-1)with concomitant accumulation of the DNA - adenylate intermediate when Mg(2+)is used as the metal cofactor. The ligation rates of matched substrates vary by up to 4-fold, but exhibit a general trend of T/A < or = G/C < C/G < A/T on both the 3'- and 5'-side of the nick. Consistent with previous studies on Thermus ligases, this Aquifex ligase exhibits greater discrimination against a mismatched base pair on the 3'-side of the nick junction. The requirement of 3' complementarity for a ligation reaction is reaffirmed by results from 1 nt insertions on either the 3'- or 5'-side of the nick. Furthermore, most of the unligatable 3' mismatched base pairs prohibit formation of the DNA-adenylate intermediate, indicating that the substrate adenylation step is also a control point for ligation fidelity. Unlike previously studied ATP ligases, gapped substrates cannot be ligated and intermediate accumulation is minimal, suggesting that complete elimination of base pair complementarity on one side of the nick affects substrate adenylation on the 5'-side of the nick junction. Relationships among metal cofactors, ligation products and intermediate, and ligation fidelity are discussed.
PMID: 10684941, UI: 20150261
Nucleic Acids Res 1999 Dec 15;27(24):e41
Microsatellite repeats consisting of dinucleotide sequences are ubiquitous in the human genome and have proven useful for linkage analysis, positional cloning and forensic identification purposes. In this study, the potential of utilizing the ligase detection reaction for the analysis of such microsatellite repeat sequences was investigated. Initially, the fidelity of thermostable DNA ligases was measured for model dinucleotide repeat sequences. Subsequently, the effect of modified oligonucleotides on ligation fidelity for dinucleotide repeats was determined using the nucleoside analogs nitroimidazole, inosine, 7-deazaguanosine and 2-pyrimidinone, as well as natural base mismatches. The measured error rates for a standard dinucleotide template indicated that the nitroimidazole nucleoside analogs could be used to increase the fidelity of ligation when compared to unmodified primers. Furthermore, use of formamide in the ligation buffer also increased ligation fidelity for dinucleotide repeat sequences. Using ligation-based assays to detect polymorphic alleles of microsatellite repeats in the human genome opens the possibility of using array-based typing of these loci for human identification, loss-of-heterozygosity studies and linkage analysis.
PMID: 10572193, UI: 20042521
Nucleic Acids Res 1999 Dec 15;27(24):e40
Up to 15% of all colorectal cancers are considered to be replication error positive (RER(+)) and contain mutations at hundreds of thousands of microsatellite repeat sequences. Recently, a number of intragenic mononucleotide repeat sequences have been demonstrated to be targets for inactivating genes in RER(+)colorectal tumors. In this study, thermostable DNA ligases were tested for the ability to detect alterations in microsatellite sequences in colon tumor samples. Ligation profiles on mononucleotide repeat sequences were determined for four related thermostable DNA ligases, Thermus thermophilus ( Tth ) ligase, Thermus sp. AK16D ligase, Aquifex aeolicus ligase and the K294R mutant of the Tth ligase. While the limit of detection for point mutations was one mutation in 1000 wild-type sequences, the ability to detect a single base deletion in a 10 base mononucleotide repeat was one mutation in 100 wild-type sequences. Furthermore, the misligation error increased exponentially as the length of the mono-nucleotide repeat increased, and was 10% of the correct signal for a 19 base mononucleotide repeat. A fluorescent ligase-based assay [polymerase chain reaction/ligase detection reaction (PCR/LDR)] correlated with results obtained using a radioactive assay to detect instability within the TGF-beta Type II receptor gene. PCR/LDR was also used to detect the APCI1307K mononucleotide repeat allele which has a carrier frequency of 6.1% in Ashkenazi Jewish individuals. In a blind study, 30 samples that had been typed for the presence of the APCI1307K allele were tested. The PCR/LDR results correlated with those obtained using sequencing and allele-specific oligonucleotide hybridization for 16 samples carrying the mutation and 13 wild-type samples. Ligation assays that characterize mononucleotide repeats can be used to rapidly detect somatic mutations in tumors, and to screen for individuals who have a hereditary predisposition to develop colon cancer.
PMID: 10572192, UI: 20042520
J Mol Biol 1999 Sep 17;292(2):251-62
Cancers arise from the accumulation of multiple mutations in genes regulating cellular growth and differentiation. Identification of such mutations in numerous genes represents a significant challenge in genetic analysis, particularly when the majority of DNA in a tumor sample is from wild-type stroma. To overcome these difficulties, we have developed a new type of DNA microchip that combines polymerase chain reaction/ligase detection reaction (PCR/LDR) with "zip-code" hybridization. Suitably designed allele-specific LDR primers become covalently ligated to adjacent fluorescently labeled primers if and only if a mutation is present. The allele-specific LDR primers contain on their 5'-ends "zip-code complements" that are used to direct LDR products to specific zip-code addresses attached covalently to a three-dimensional gel-matrix array. Since zip-codes have no homology to either the target sequence or to other sequences in the genome, false signals due to mismatch hybridizations are not detected. The zip-code sequences remain constant and their complements can be appended to any set of LDR primers, making our zip-code arrays universal. Using the K- ras gene as a model system, multiplex PCR/LDR followed by hybridization to prototype 3x3 zip-code arrays correctly identified all mutations in tumor and cell line DNA. Mutations present at less than one per cent of the wild-type DNA level could be distinguished. Universal arrays may be used to rapidly detect low abundance mutations in any gene of interest. Copyright 1999 Academic Press.
PMID: 10493873, UI: 99425309
Clin Biochem 1999 Jun;32(4):287-90
PMID: 10463822, UI: 99391479
Nucleic Acids Res 1999 Apr 15;27(8):1819-27
A high sensitivity method for detecting low level mutations is under development. A PCR reaction is performed in which a restriction site is introduced in wild-type DNA by alteration of specific bases. Digestion of wild-type DNA by the cognate restriction endonuclease (RE) enriches for products with mutations within the recognition site. After reamplification, mutations are identified by a ligation detection reaction (LDR). This PCR/RE/LDR assay was initially used to detect PCR error in known wild-type samples. PCR error was measured in low |Deltap K a| buffers containing tricine, EPPS and citrate, as well as otherwise identical buffers containing Tris. PCR conditions were optimized to minimize PCR error using perfect match primers at the Msp I site in the p53 tumor suppressor gene at codon 248. However, since mutations do not always occur within pre-existing restriction sites, a generalized PCR/RE/LDR method requires the introduction of a new restriction site. In principle, PCR with mismatch primers can alter specific bases in a sequence and generate a new restriction site. However, extension from 3' mismatch primers may generate misextension products. We tested conversion of the Msp I (CCGG) site to a Taq I site (TCGA). Conversion was unsuccessful using a natural base T mismatch primer set. Conversion was successful when modified primers containing the 6 H,8 H -3, 4-dihydropyrimido[4,5- c ][1,2]oxazine-7-one (Q6) base at 3'-ends were used in three cycles of preconversion PCR prior to conversion PCR using the 3' natural base T primers. The ability of the pyrimidine analog Q6 to access both a T-like and C-like tautomer appears to greatly facilitate the conversion.
PMID: 10101189, UI: 99207121
Nucleic Acids Res 1999 Apr 15;27(8):1810-8
We compared the efficiency of PCR amplification using primers containing either a nucleotide analog or a mismatch at the 3' base. To determine the distribution of bases inserted opposite eight different analogs, 3' analog primers were used to amplify four different templates. The products from the reactions with the highest amplification efficiency were sequenced.Analogs allowing efficient amplification followed by insertion of a new base at that position are herein termed 'convertides'. The three convertides with the highest amplification efficiency were used to convert sequences containing C, T, G and A bases into products containing the respective three remaining bases. Nine templates were used to generate conversion products, as well as non-conversion control products with no base change. We compared the ability of natural bases to convert specific sites with and without a preconversion step using nucleotide analog primers. Conversion products were identified by a ligation detection reaction using primers specific for the converted sequence. We found that conversions resulting in transitions were easier to accomplish than transversions and that sequence context influences conversion. Specifically, primer slippage appears to be an important mechanism for producing artifacts via polymerase extension of a 3' base or analog transiently base paired to neighboring bases of the template. Nucleotide analogs could often reduce conversion artifacts and increase the yield of the expected product. While new analogs are needed to reliably achieve transversions, the current set have proven effective for creating transition conversions.
PMID: 10101188, UI: 99207120
Oncogene 1999 Jan 7;18(1):27-38
Point mutations in codons 12, 13, and 61 of the K-ras gene occur early in the development of colorectal cancer and are preserved throughout the course of tumor progression. These mutations can serve as biomarkers for shed or circulating tumor cells and may be useful for diagnosis of early, curable tumors and for staging of advanced cancers. We have developed a multiplex polymerase chain reaction/ligase detection reaction (PCR/LDR) method which identifies all 19 possible single-base mutations in K-ras codons 12, 13, and 61, with a sensitivity of 1 in 500 wild-type sequences. In a blinded study, 144 paraffin-embedded archival colon carcinomas were microdissected and K-ras mutations determined by both dideoxy-sequencing and multiplex PCR/LDR. Results were concordant for 134 samples. The ten discordant samples were re-evaluated using higher sensitivity uniplex PCR/LDR, and the original multiplex PCR/LDR result was confirmed in nine of these ten cases. Multiplex PCR/LDR was able to identify mutations in solid tumors or paraffin-embedded tissues containing a majority of wild-type stromal cells, with or without microdissection. The technique is well suited for large scale studies and for analysis of clinical samples containing a minority population of mutated cells.
PMID: 9926917, UI: 99124107
Nucleic Acids Res 1999 Feb 1;27(3):788-94
NAD+-dependent DNA ligases from thermophilic bacteria Thermus species are highly homologous with amino acid sequence identities ranging from 85 to 98%. Thermus species AK16D ligase, the most divergent of the seven Thermus isolates collected worldwide, was cloned, expressed in Escherichia coli and purified to homogeneity. This Thermus ligase is similar to Thermus thermophilus HB8 ligase with respect to pH, salt, NAD+, divalent cation profiles and steady-state kinetics.However, the former is more discriminative toward T/G mismatches at the 3'-side of the ligation junction, as judged by the ratios of initial ligation rates of matched and mismatched substrates. The two wild-type Thermus ligases and a Tth ligase mutant (K294R) demonstrate 1-2 orders of magnitude higher fidelity than viral T4 DNA ligase. Both Thermus ligases are active with either the metal cofactor Mg2+, Mn2+or Ca2+but not with Co2+, Ni2+, Cu2+or Zn2+. While the nick closure step with Ca2+becomes rate-limiting which results in the accumulation of DNA-adenylate intermediate, Ni2+only supports intermediate formation to a limited extent. Both Thermus ligases exhibit enhanced mismatch ligation when Mn2+is substituted for Mg2+, but the Tsp. AK16D ligase remains more specific toward perfectly matched substrate.
PMID: 9889274, UI: 99108143
J Biol Chem 1998 Dec 4;273(49):33002-10
Metal cofactors (Mg2+ and Mn2+) modulate both specific DNA binding and strand cleavage in the TaqI endonuclease (Cao, W., Mayer, A. N., and Barany, F. (1995) Biochemistry 34, 2276-2283). This work attempts to establish the structural basis of TaqI-DNA-metal2+ interactions using an affinity cleavage technique. The protein was cleaved by localized hydroxyl radicals generated by oxidizing Fe2+ within the metal binding sites. Cleavage fragments were separated by SDS-polyacrylamide gel electrophoresis, and cleavage sites were determined using micropeptide sequencing. Eleven amino acid residues in the vicinity of cleavage sites were selected for site-directed mutagenesis. The negative charge at Asp137 is essential for DNA cleavage but not required for sequence specific binding. Mutations at Asp142 abolish both specific binding and catalysis, except for D142E, which converts TaqI into a completely Mn2+-dependent endonuclease. The positive charge at Lys158 appears to be important for both specific binding and catalysis. Mutations at other sites affect binding and/or catalysis to different degrees, except Trp113 and Glu135, which appear to be nonessential for the TaqI enzyme activity. The critical residues for TaqI function are distinct from the PDX14-20(E/D)XK catalytic motif elucidated from other endonucleases.
PMID: 9830053, UI: 99047690
Biochem J 1998 Jul 15;333 ( Pt 2):425-31
Two TaqI endonuclease (hereafter referred to as TaqI) isoschizomer genes, tsp32IR from Thermus species SM32 of Azores and tfiTok6A1I from T. filiformis Tok6A1 of New Zealand, were cloned in Escherichia coli. The overexpressed enzymes were partly purified and their thermostability was determined. In the medium-salt buffer, Tsp32IR, TfiTok6A1I and one previously cloned TaqI isoschizomer (TthHB8I) were more thermostable than TaqI. Tsp32IR remained partly active up to 90 degreesC in the low-salt buffer. Six amino acid residues that are identical in the three high thermostability isoschizomers (Tsp32IR, TfiTok6A1I and TthHB8I) but differ in TaqI might provide added rigidity for thermostabilization. These include four proline residues located in or near loop regions, and one alanine and one arginine located at helix regions in the predicted TaqI endonuclease secondary structure. The possible role of these residues in thermostabilization was evaluated by mutagenizing the TaqI enzyme. Mutants generated at these six positions were less thermostable than wild-type TaqI. The results suggest that the surrounding sequence or structural context might be as important as the mutation itself.
PMID: 9657984, UI: 98324905
Gene 1997 Sep 15;197(1-2):205-14
Eight TaqI isoschizomer genes, two from Yellowstone National Park, one from Japan, two from New Zealand, two from Portugal, and one from the Azores (1000 miles west of Portugal), were PCR-amplified and sequenced. Sequence alignment of isoschizomers isolated from close geographical locations shows identical or almost identical protein sequences, while isoschizomers from distant sites demonstrate considerable diversity, ranging from 54 to 75% in amino acid identity. Accordingly, these isoschizomers were arranged into four geographical groups, i.e., USA as represented by Thermus aquaticus YT1, Japan by Thermus thermophilus HB8, New Zealand by Thermus filiformis Tok6A1, Portugal by Thermus sp. SM32. The complete ORFs of two new representative genes, tfiTok6A1I and tsp32IR, were obtained by bubble PCR. Unlike M . TaqI-R.TaqI and M . TthHB8I-R . TthHB8I which exhibit an unusual 13-codon overlap, the methylase and endonuclease genes are each separated by 15 nucleotides in the TfiTok6A1I and Tsp32IR restriction-modification systems. Phylogenetic analysis suggests that initially TfiTok6A1I diverged from a common ancestor, then Tsp32IR branched out, and finally TaqI and TthHB8I diverged from each other during evolution.
PMID: 9332368, UI: 97473514
Hum Mol Genet 1996 Dec;5(12):2039-48
Steroid 21-hydroxylase deficiency is among the most common inborn errors of metabolism in man. Characterization of mutations in the 21-hydroxylase gene (CYP21) has permitted genetic diagnosis, facilitated by the polymerase chain reaction (PCR). The most common mutation is conversion of an A or C at nt656 to a G in the second intron causing aberrant splicing of mRNA. Homozygosity for nt656G is associated with profoundly deficient adrenal cortisol and aldosterone synthesis, secondary hypersecretion of adrenal androgens, and a severe form of congenital adrenal hyperplasia (CAH) characterized by ambiguous genitalia and/or sodium wasting in newborns. During the course of genetic analysis of CYP21 mutations in CAH families, we and others have noticed a number of relatives genotyped as nt656G homozygotes, yet showing no clinical signs of disease. A number of lines of evidence have led us to propose that the putative asymptomatic nt656G/G individuals are incorrectly typed due to dropout of one haplotype during PCR amplification of CYP21. For prenatal diagnosis, we recommend that microsatellite typing be used as a supplement to CYP21 genotyping in order to resolve ambiguities at nt656.
PMID: 8968761, UI: 97123514
Nucleic Acids Res 1996 Aug 1;24(15):3079-85
DNA ligases play a pivotal role in DNA replication, repair and recombination. Reactions catalyzed by DNA ligases consist of three steps: adenylation of the ligase in the presence of ATP or NAD+, transferring the adenylate moiety to the 5'-phosphate of the nicked DNA substrate (deadenylation) and sealing the nick through the formation of a phosphodiester bond. Thermus thermophilus HB8 DNA ligase (Tth DNA ligase) differs from mesophilic ATP-dependent DNA ligases in three ways: (i) it is NAD+ dependent; (ii) its optimal temperature is 65 instead of 37 degrees C; (iii) it has higher fidelity than T4 DNA ligase. In order to understand the structural basis underlying the reaction mechanism of Tth DNA ligase, we performed site-directed mutagenesis studies on nine selected amino acid residues that are highly conserved in bacterial DNA ligases. Examination of these site-specific mutants revealed that: residue K118 plays an essential role in the adenylation step; residue D120 may facilitate the deadenylation step; residues G339 and C433 may be involved in formation of the phosphodiester bond. This evidence indicates that a previously identified KXDG motif for adenylation of eukaryotic DNA ligases [Tomkinson, A.E., Totty, N.F., Ginsburg, M. and Lindahl, T. (1991) Proc. Natl. Acad. Sci. USA, 88, 400-404] is also the adenylation site for NAD+-dependent bacterial DNA ligases. In a companion paper, we demonstrate that mutations at a different Lys residue, K294, may modulate the fidelity of Tth DNA ligase.
PMID: 8760897, UI: 96357146
Nucleic Acids Res 1996 Aug 1;24(15):3071-8
The DNA ligase from Thermus thermophilus (Tth DNA ligase) seals single-strand breaks (nicks) in DNA duplex substrates. The specificity and thermostability of this enzyme are exploited in the ligase chain reaction (LCR) and ligase detection reaction (LDR) to distinguish single base mutations associated with genetic diseases. Herein, we describe a quantitative assay using fluorescently labeled substrates to study the fidelity of Tth DNA ligase. The enzyme exhibits significantly greater discrimination against all single base mismatches on the 3'-side of the nick in comparison with those on the 5'-side of the nick. Among all 12 possible single base pair mismatches on the 3'-side of the nick, only T-G and G-T mismatches generated a quantifiable level of ligation products after 23 h incubation. The high fidelity of Tth DNA ligase can be improved further by introducing a mismatched base or a universal nucleoside analog at the third position of the discriminating oligonucleotide. Finally, two mutant Tth DNA ligases, K294R and K294P, were found to have increased fidelity using this assay.
PMID: 8760896, UI: 96357145
Genomics 1995 Sep 1;29(1):152-62
Steroid 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia, an inherited inability to synthesize cortisol that occurs in 1 in 10,000-15,000 births. Affected females are born with ambiguous genitalia, a condition that can be ameliorated by administering dexamethasone to the mother for most of gestation. Prenatal diagnosis is required for accurate treatment of affected females as well as for genetic counseling purposes. Approximately 95% of mutations causing this disorder result from recombinations between the gene encoding the 21-hydroxylase enzyme (CYP21) and a linked, highly homologous pseudogene (CYP21P). Approximately 20% of these mutations are gene deletions, and the remainder are gene conversions that transfer any of nine deleterious mutations from the CYP21P pseudogene to CYP21. We describe a methodology for genetic diagnosis of 21-hydroxylase deficiency that utilizes gene-specific PCR amplification in conjunction with thermostable DNA ligase to discriminate single nucleotide variations in a multiplexed ligation detection assay. The assay has been designed to be used with either fluorescent or radioactive detection of ligation products by electrophoresis on denaturing acrylamide gels and is readily adaptable for use in other disease systems.
PMID: 8530065, UI: 96079103