PCR-free mutation detection of BRCA1 on a zip-code microarray using ligase chain reaction

We describe here ligation-based strategy to detect mutations in BRCA1 utilizing zip-code microarray technology. In our first approach, PCR was performed to amplify the genomic regions containing the mutation sites. The PCR products were then used as templates in a subsequent ligation reaction using two ligation primers that flanked the mutation site. The primary allele-specific primer is designed to contain a base of mutation site at its 3′ end with 5′ complementarity to the respective zip-code sequence while the secondary common primer is modified by biotin at its 3′ end. Depending on the genotype of samples at the mutation site, the nick between the two ligation primers can be sealed in the presence of DNA ligase. The ligation products were then hybridized on the zip-code microarray followed by staining with streptavidine-cy3 to generate a fluorescent signal. Using this strategy we successfully genotyped selected Korean-specific mutation sites in exon 11 of BRCA1 with a wild type and two heterozygote mutant samples. Furthermore, we also demonstrated that ligase chain reaction using unamplified genomic DNA as direct templates is enough to generate sufficient signals for correct genotypings in a multiplexed manner, verifying first that PCR is not essential for this microarray-based strategy.     

Analysis of point mutations of the BRCA1 gene by hybridization with hydrogel microarrays

BRCA1 mutations are associated with a higher risk of breast (BC) and ovarian cancer in women. Testing for such mutations allows BC prognosis, selection of an individual treatment strategy, and prevention of disease recurrence. Hybridization on a hydrogel microarray was developed for identifying point mutations in BRCA1. The microarray was designed to detect five-point mutations: 185delAG, 300T→G, 4153delA, 4158A→G, and 5382insC. The microarray was tested with 36 control specimens with known genotypes and used to examine 65 BC patients. The results demonstrated the advantage of employing the microarray in analyzing BRCA1 mutations.     

Evaluation and optimisation of indel detection workflows for ion torrent sequencing of the BRCA1 and BRCA2 genes

Background

The Ion Torrent PGM is a popular benchtop sequencer that shows promise in replacing conventional Sanger sequencing as the gold standard for mutation detection. Despite the PGM’s reported high accuracy in calling single nucleotide variations, it tends to generate many false positive calls in detecting insertions and deletions (indels), which may hinder its utility for clinical genetic testing.

Results

Recently, the proprietary analytical workflow for the Ion Torrent sequencer, Torrent Suite (TS), underwent a series of upgrades. We evaluated three major upgrades of TS by calling indels in the BRCA1 and BRCA2 genes. Our analysis revealed that false negative indels could be generated by TS under both default calling parameters and parameters adjusted for maximum sensitivity. However, indel calling with the same data using the open source variant callers, GATK and SAMtools showed that false negatives could be minimised with the use of appropriate bioinformatics analysis. Furthermore, we identified two variant calling measures, Quality-by-Depth (QD) and VARiation of the Width of gaps and inserts (VARW), which substantially reduced false positive indels, including non-homopolymer associated errors without compromising sensitivity. In our best case scenario that involved the TMAP aligner and SAMtools, we achieved 100% sensitivity, 99.99% specificity and 29% False Discovery Rate (FDR) in indel calling from all 23 samples, which is a good performance for mutation screening using PGM.

Conclusions

New versions of TS, BWA and GATK have shown improvements in indel calling sensitivity and specificity over their older counterpart. However, the variant caller of TS exhibits a lower sensitivity than GATK and SAMtools. Our findings demonstrate that although indel calling from PGM sequences may appear to be noisy at first glance, proper computational indel calling analysis is able to maximize both the sensitivity and specificity at the single base level, paving the way for the usage of this technology for future clinical genetic testing.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-516) contains supplementary material, which is available to authorized users.     

Development of a protein microarray using sequence-specific DNA binding domain on DNA chip surface

A protein microarray based on DNA microarray platform was developed to identify protein-protein interactions in vitro. The conventional DNA chip surface by 156-bp PCR product was prepared for a substrate of protein microarray. High-affinity sequence-specific DNA binding domain, GAL4 DNA binding domain, was introduced to the protein microarray as fusion partner of a target model protein, enhanced green fluorescent protein. The target protein was oriented immobilized directly on the DNA chip surface. Finally, monoclonal antibody of the target protein was used to identify the immobilized protein on the surface. This study shows that the conventional DNA chip can be used to make a protein microarray directly, and this novel protein microarray can be applicable as a tool for identifying protein-protein interactions.     

BRCA1和BRCA2与DNA损伤修复及转录调控

乳腺癌和卵巢癌敏感基因BRCA1和BRCA2与同源重组,DNA损伤修复,胚胎生长,转录调控及遍在蛋白化有关,其中,BRCA1和BRCA2在DNA损伤修复和转录调控中功能的确定,将有助于探讨和阐明两者的肿瘤抑制功能及其机理,作者将综述近年来有关BRCA1和BRCA2在DNA损伤修复和转录调控中功能研究的最新进展。     

Mutation detection in the breast cancer gene BRCA1 using the protein truncation test

About 400 distinct mutations have been defined in the BRCA1 gene, and these are spread fairly evenly through the 5592 bp of coding DNA. This circumstance presents a formidable challenge for mutation screening. Apart from total direct sequencing, the preferred screening method has been single-strand conformation polymorphism (SSCP) gels, with a smaller input from constant denaturant gradient electrophoresis (CDGE), heteroduplex (HD) analysis, and mismatch cleavage. The protein truncation test (PTT) was used early in BRCA1 mutation screening but has not been widely adopted, perhaps because a straightforward analysis of the whole BRCA1 gene requires working with RNA and all its perceived problems. The present work was undertaken to assess the practicality of using the PTT under routine conditions for the screening of long genes such as BRCA1 that are not highly expressed in lymphocytes. We conclude that, provided RNA preparation is carried out effectively and consistently, the PTT approach has significant advantages over other methodologies such as SSCP gels.     

乳腺癌易感蛋白1在DNA损伤修复中的作用

人类乳腺癌易感基因1(breast cancer susceptibility gene 1,BRCA1)首先是在乳腺癌家族中发现的,是具有遗传倾向的乳腺癌和卵巢癌易感基因,其基因的突变与家族性乳腺癌及卵巢癌的发生有密切联系。BRCA1是一种抑癌基因,其基因产物可以参与维持基因组稳定性的多条细胞信号通路,例如DNA损伤诱导的细胞周期调控、DNA损伤修复、基因转录调节、细胞凋亡、泛素化等重要的细胞活动。本文就近几年来BRCA1在DNA损伤修复中的作用的研究进展作一综述,包括DNA损伤诱导的细胞周期检查点的激活和DNA损伤修复两方面。     

Oligo-chip based detection of tick-borne bacteria

We have developed an oligonucleotide-chip based assay for detection of 16S ribosomal PCR products from tick-borne bacteria. This chip contains 14 specific probes, which target variable regions of 16S rDNA of tick-borne bacteria including Borrellia spp., Rickettsia spp., Anaplasma spp., Coxiella burnetii and Francisella tularensis. The specificity of these probes was tested by hybridization of the chip with fluorescently labeled PCR products amplified from the genomic DNA of selected tick-borne bacteria. The assay was also tested for detection of tick-borne bacteria in single ticks.     

Label-free electrical detection of DNA hybridization for the example of influenza virus gene sequences

Microarrays based on DNA-DNA hybridization are potentially useful for detecting and subtyping viruses but require fluorescence labeling and imaging equipment. We investigated a label-free electrical detection system using electrochemical impedance spectroscopy that is able to detect hybridization of DNA target sequences derived from avian H5N1 influenza virus to gold surface-attached single-stranded DNA oligonucleotide probes. A 23-nt probe is able to detect a 120-nt base fragment of the influenza A hemagglutinin gene sequence. We describe a novel method of data analysis that is compatible with automatic measurement without operator input, contrary to curve fitting used in conventional electrochemical impedance spectroscopy (EIS) data analysis. A systematic investigation of the detection signal for various spacer molecules between the oligonucleotide probe and the gold surface revealed that the signal/background ratio improves as the length of the spacer increases, with a 12- to 18-atom spacer element being optimal. The optimal spacer molecule allows a detection limit between 30 and 100 fmol DNA with a macroscopic gold disc electrode of 1 mm radius. The dependence of the detection signal on the concentration of a 23-nt target follows a binding curve with an approximate 1:1 stoichiometry and a dissociation constant of KD=13+/-4 nM at 295 K.     

Identification and functional characterization of a PP1-binding site in BRCA1

The phosphorylation state of the tumor suppressor protein BRCA1 is tightly associated with its functions including cell cycle control and DNA repair. Protein kinases involved in the DNA damage checkpoint control, such as ATM, ATR, and hCds1/Chk2, have been shown to phosphorylate and activate BRCA1 upon DNA damage. We reported previously that protein phosphatase 1alpha (PP1alpha) interacts with and dephosphorylates hCds1/Chk2-phosphorylated BRCA1. This study demonstrates the identification of a PP1-binding motif 898KVTF901 in BRCA1. Mutation or deletion of critical residues in this PP1-binding motif substantially reduces the interaction between BRCA1 and PP1alpha. PP1alpha can also dephosphorylate ATM and ATR phosphorylation sites in BRCA1 and may serve as a general regulator for BRCA1 phosphorylation. Unlike wild-type BRCA1, expression of the PP1 non-binding mutant BRCA1 protein in BRCA1-deficient cells failed to enhance survival after DNA damage. Taken together, these results suggest that interaction with PP1alpha is important for BRCA1 function.     

Centrosomal BRCA2 is a target protein of membrane type-1 matrix metalloproteinase (MT1-MMP)

BRCA2 localizes to centrosomes between G1 and prophase and is removed from the centrosomes during mitosis, but the underlying mechanism is not clear. Here we show that BRCA2 is cleaved into two fragments by membrane type-1 matrix metalloproteinase (MT1-MMP), and that knockdown of MT1-MMP prevents the removal of BRCA2 from centrosomes during metaphase. Mass spectrometry mapping revealed that the MT1-MMP cleavage site of human BRCA2 is between Asn-2135 and Leu-2136 (²¹³²LSNN/LNVEGG²¹⁴¹), and the point mutation L2136D abrogated MT1-MMP cleavage. Our data demonstrate that MT1-MMP proteolysis of BRCA2 regulates the abundance of BRCA2 on centrosomes.     

Visual detection of single-stranded target DNA using pyrroloquinoline-quinone-loaded liposomes as a tracer

The preparation of DNA-tagged liposomes containing an encapsulated prosthetic group tracer, pyrroloquinoline quinone (PQQ), and their application to the development of a sandwich-type hybridization assay for the visual detection of single-stranded DNA are described. Capture DNA is conjugated to the surface of microtiter plate wells through a biotin-streptavidin interaction. Target DNA is incubated with the plate in high salt concentrations. The reporter DNA-tagged liposomes encapsulating PQQ, the prosthetic group of the apo-enzyme glucose dehydrogenase (GDH), are used as the label to probe for bound target DNA. After washing away unbound liposomes and subsequent lysis of the bound fraction by surfactant, PQQ is released and available to activate the apo-enzyme. In the presence of glucose and a redox dye, 2,6-dichlorophenol indophenol (DCPIP), the dye is reduced to yield an optical color change from blue to colorless. This transition is observed visually or spectrophotometrically. The degree of optical change is proportional to the amount of PQQ present, which directly relates to the number of liposomes and, thus, the total amount of target DNA. An arbitrary target DNA sequence is used as a model system, and a limit of detection of 62 fmol is achieved.     

Ferrocene conjugated oligonucleotide for electrochemical detection of DNA base mismatch

We describe the synthesis, binding, and electrochemical properties of ferrocene-conjugated oligonucleotides (Fc-oligos). The key step for the preparation of Fc-oligos contains the coupling of vinylferrocene to 5-iododeoxyuridine via Heck reaction. The Fc-conjugated deoxyuridine phosphoramidite was used in the Fc-oligonucleotide synthesis. We show that thiol-modified Fc-oligos deposited onto gold electrodes possess potential ability in electrochemical detection of DNA base mismatch.     

Comparative reactivity of mismatched and unpaired bases in relation to their type and surroundings. Chemical cleavage of DNA mismatches in mutation detection analysis

Systematic study of chemical reactivity of non-Watson–Crick base pairs depending on their type and microenvironment was performed on a model system that represents two sets of synthetic DNA duplexes with all types of mismatched and unmatched bases flanked by T·A or G·C pairs. Using comparative cleavage pattern analysis, we identified the main and additional target bases and performed quantitative study of the time course and efficacy of DNA modification caused by potassium permanganate or hydroxylamine. Potassium permanganate in combination with tetraethylammonium chloride was shown to induce DNA cleavage at all mismatched or bulged T residues, as well as at thymines of neighboring canonical pairs. Other mispaired (bulged) bases and thymine residues located on the second position from the mismatch site were not the targets for KMnO₄ attack. In contrast, hydroxylamine cleaved only heteroduplexes containing mismatched or unmatched C residues, and did not modify adjacent cytosines. However when G·C pairs flank bulged C residue, neighboring cytosines are also attacked by hydroxylamine due to defect migration. Chemical reactivity of target bases was shown to correlate strongly with the local disturbance of DNA double helix at mismatch or bulge site. With our model system, we were able to prove the absence of false-negative and false-positive results. Portion of heteroduplex reliably revealed in a mixture with corresponding homoduplex consists of 5% for bulge bases and “open” non-canonical pairs, and 10% for wobble base pairs giving minimal violations in DNA structure. This study provides a complete understanding of the principles of mutation detection methodology based on chemical cleavage of mismatches and clarifies the advantages and limitations of this approach in various biological and conformational studies of DNA.     

Hydroxyethylcellulose as an effective polymer network for DNA analysis in uncoated glass microchips: optimization and application to mutation detection via heteroduplex analysis

The nature of the sieving matrix for DNA fragment separation is of immense importance in capillary and microchip electrophoresis. The chemical nature of the surface of the capillary or microchannel wall is equally as important, particularly with DNA electrophoresis where a substantial electroosmotic flow (EOF) may be detrimental to the separation. Although DNA analysis has been carried out successfully in both coated and uncoated capillaries, analysis of unpurified polymerase chain reaction products has been carried out primarily with covalently coated surfaces, especially with microchip electrophoresis. In this report, double-stranded (ds) DNA fragment analysis using hydroxyethylcellulose (HEC) buffered in 1xTris-borate-EDTA is demonstrated both in uncoated capillaries and in microchips. EOF was suppressed 20% in the presence of 1.5% HEC, and the effectiveness of HEC as a polymer for dsDNA fragment analysis was dependent on the pH, with pH 8.6 being optimal. Using separation efficiency (number of theoretical plates) and resolution to gauge the effectiveness of a variety of polymers for the capillary separation of dsDNA fragments in the size range 60-587bp, HEC was found to be comparable in performance to polydimethylacrylamide (PDMA), and superior to linear polyacrylamide and polyethylene oxide for DNA analysis. With respect to longevity and robust performance, HEC could be used effectively in an uncoated capillary for more than 40 runs and for more than 90 runs (without replenishing the polymer) in an uncoated microchip. Application of the optimized HEC conditions is demonstrated through its ability to facilitate heteroduplex analysis.