Chemistry of locked nucleic acids (LNA): Design, synthesis, and bio-physical properties

Preparation of LNA nucleosides requires a number of synthetic steps but very efficient procedures have been developed, as have protocols for synthesis of LNA oligonucleotides on automated DNA synthesizers. In all cases, LNA oligonucleotides have exhibited good aqueous solubility as would be expected from their close structural resemblance to the natural nucleic acids. The universality of LNA mediated high-affinity and specific hybridization has been demonstrated extensively with a large number of duplex forming LNA-oligonucleotides. Most importantly, most of the members of the LNA molecular family have been shown to exert their substantial affinity increase (i) in combination with standard DNA, RNA and contemporary analogues and (ii) whether inserted as single nucleosides in an oligonucleotide or as blocks of contiguous nucleotides, an important point. The works on TFO's is expanding the usefulness of LNA to double strand recognition and it has been demonstrated that LNA it is a promising structure for further base modifications in the pursuit of global sequence specific recognition of DNA.     

Chemistry of locked nucleic acids (LNA): Design, synthesis, and bio-physical properties

Summary Preparation of LNA nucleosides requires a number of synthetic steps but very efficient procedures have been developed, as have protocols for synthesis of LNA oligonucleotides on automated DNA synthesizers. In all cases, LNA oligonucleotides have exhibited good aqueous solubility as would be expected from their close structural resemblance to the natural nucleic acids. The universality of LNA mediated high-affinity and specific hybridization has been demonstrated extensively with a large number of duplex forming LNA-oligonucleotides. Most importantly, most of the members of the LNA molecular family have been shown to exert their substantial affinity increase (i) in combination with standard DNA, RNA and contemporary analogues and (ii) whether inserted as single nucleosides in an oligonucleotide or as blocks of contiguous nucleotides, an important point. The works on TFO's is expanding the usefulness of LNA to double strand recognition and it has been demonstrated that LNA it is a promising structure for further base modifications in the pursuit of global sequence specific recognition of DNA.     

Chemistry of locked nucleic acids (LNA): Design, synthesis, and bio-physical properties

Summary Preparation of LNA nucleosides requires a number of synthetic steps but very efficient procedures have been developed, as have protocols for synthesis of LNA oligonucleotides on automated DNA synthesizers. In all cases, LNA oligonucleotides have exhibited good aqueous solubility as would be expected from their close structural resemblance to the natural nucleic acids. The universality of LNA mediated high-affinity and specific hybridization has been demonstrated extensively with a large number of duplex forming LNA-oligonucleotides. Most importantly, most of the members of the LNA molecular family have been shown to exert their substantial affinity increase (i) in combination with standard DNA, RNA and contemporary analogues and (ii) whether inserted as single nucleosides in an oligonucleotide or as blocks of contiguous nucleotides, an important point. The works on TFOs is expanding the usefulness of LNA to double strand recognition and it has been demonstrated that LNA it is a promising structure for further base modifications in the pursuit of global sequence specific recognition of DNA.     

Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis

Drug-resistant Mycobacterium tuberculosis can be rapidly diagnosed through nucleic acid amplification techniques by analyzing the variations in the associated gene sequences. In the present study, a locked nucleic acid (LNA) probe-based real-time PCR assay was developed to identify the mutations in the rpoB gene associated with rifampin (RFP) resistance in M. tuberculosis. Six LNA probes with the discrimination capability of one-base mismatch were designed to monitor the 23 most frequent rpoB mutations. The target mutations were identified using the probes in a “probe dropout” manner (quantification cycle = 0); thus, the proposed technique exhibited superiority in mutation detection. The LNA probe-based real-time PCR assay was developed in a two-tube format with three LNA probes and one internal amplification control probe in each tube. The assay showed excellent specificity to M. tuberculosis with or without RFP resistance by evaluating 12 strains of common non-tuberculosis mycobacteria. The limit of detection of M. tuberculosis was 10 genomic equivalents (GE)/reaction by further introducing a nested PCR method. In a blind validation of 154 clinical mycobacterium isolates, 142/142 (100%) were correctly detected through the assay. Of these isolates, 88/88 (100%) were determined as RFP susceptible and 52/54 (96.3%) were characterized as RFP resistant. Two unrecognized RFP-resistant strains were sequenced and were found to contain mutations outside the range of the 23 mutation targets. In conclusion, this study established a sensitive, accurate, and low-cost LNA probe-based assay suitable for a four-multiplexing real-time PCR instrument. The proposed method can be used to diagnose RFP-resistant tuberculosis in clinical laboratories.     

Novel Methodology for Rapid Detection of KRAS Mutation Using PNA-LNA Mediated Loop-Mediated Isothermal Amplification

Detecting point mutation of human cancer cells quickly and accurately is gaining in importance for pathological diagnosis and choice of therapeutic approach. In the present study, we present novel methodology, peptide nucleic acid—locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA mediated LAMP), for rapid detection of KRAS mutation using advantages of both artificial DNA and LAMP. PNA-LNA mediated LAMP reactions occurred under isothermal temperature conditions of with 4 primary primers set for the target regions on the KRAS gene, clamping PNA probe that was complimentary to the wild type sequence and LNA primers complementary to the mutated sequences. PNA-LNA mediated LAMP was applied for cDNA from 4 kinds of pancreatic carcinoma cell lines with or without KRAS point mutation. The amplified DNA products were verified by naked-eye as well as a real-time PCR equipment. By PNA-LNA mediated LAMP, amplification of wild type KRAS DNA was blocked by clamping PNA probe, whereas, mutant type KRAS DNA was significantly amplified within 50 min. Mutant alleles could be detected in samples which diluted until 0.1% of mutant-to-wild type ratio. On the other hand, mutant alleles could be reproducibly with a mutant-to-wild type ratio of 30% by direct sequencing and of 1% by PNA-clamping PCR. The limit of detection (LOD) of PNA-LNA mediated LAMP was much lower than the other conventional methods. Competition of LNA clamping primers complementary to two different subtypes (G12D and G12V) of mutant KRAS gene indicated different amplification time depend on subtypes of mutant cDNA. PNA-LNA mediated LAMP is a simple, rapid, specific and sensitive methodology for the detection of KRAS mutation.     

A LDL receptor gene homozygous mutation: PCR amplification, direct genomic sequencing, associated haplotype, rapid screening for frequency

Many mutations in the LDL receptor (LDLR) gene have now been identified mostly as gross gene rearrangements, however they only represent a weak percentage of all deleterious gene mutations causing Familial Hypercholesterolemia (FH). This discrepancy may be related to the difficulties in characterizing point or small defective mutations. In a three-generation family with Familial Hypercholesterolemia, one specific haplotype constructed with 12 intragenic restriction fragment length polymorphisms (RFLP) cosegregated with the disease, while in the consanguineous propositus there was homozygosity for this haplotype. By polymerase chain reaction (PCR) amplification followed by direct sequencing there was unequivocal evidence for a double dose of a unique mutation, (namely a duplication of 4 bases in exon 17), while there was a single dose in heterozygote relatives. We consequently screened a population selected under clinical and geographical criteria for this mutation by PCR and allele specific oligonucleotides (ASO) hybridization. None of the 158 type IIa individuals tested carried the same mutation. Herein, is a rapid combined genetic and molecular approach to characterize and evaluate the frequency of LDL Receptor gene mutations causing Familial Hypercholesterolemia, towards targeted prevention and therapy.     

Development, multiplexing, and application of ARMS tests for common mutations in the CFTR gene.

The amplification refractory mutation system (ARMS) is a simple, rapid and reliable method for the detection of any mutation involving single base changes or small deletions. We have applied ARMS methodology to the detection of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Single ARMS tests have been developed for 11 CFTR mutations found in the northwest of England. ARMS reactions for the most common mutations have been multiplexed to give a test which will detect the presence of the delta F508, G551D, G542X, and 621 + 1G----T mutations in a DNA sample. The multiplex test has been validated by the analysis of over 500 previously genotyped samples and has been found to be completely accurate. The rapid detection of the most common mutations has enabled early molecular confirmation of suspected cystic fibrosis in neonates, rapid typing of cystic fibrosis patients and their relatives, and testing of sperm and egg donors.     

On-chip oligonucleotide ligation assay using one-dimensional microfluidic beads array for the detection of low-abundant DNA point mutations

The detection of low-abundant DNA point mutations is very important for the early prediction of cancer, diagnostics of disease and clinical prognosis. In this paper, an on-chip oligonucleotide ligation approach that arrayed a series of functionalized beads in a single microfluidic channel was described for detection of low-abundant point mutations in p53 gene. This gene carried the point mutation with high diagnostic value for assessment of tumor progression and resectional borders. This work extended our prior efforts using one-dimensional (1-D) microfluidic beads array for protein and nucleic acid molecular profiling, and displayed high discrimination sensitivity to mutations detection due to the enhanced mass transport capability caused by microfluidic addressing format of beads array. As a demonstration, it was found that the on-chip beads ligation held high mutation discrimination sensitivity in 1 pM quantities at a SNR (signal-to-noise ratio) >2 using synthesized DNA oligonucleotides in accordance with target fragment. The RT-PCR products of tumor cell line A549, CNE2 and SKBr-3 were further examined to distinguish the point mutation at codon 175 of p53 gene. This approach was capable of detecting a point mutation in a p53 oncogene at a level of 1 mutant in 1000 wild-type sequences using PCR products without the need of LDR amplification. Additionally, this on-chip beads ligation approach also displayed other microfluidic-based advantages of simple handling (one sample injection per test), little reagent quantities, and low potential of contaminations.     

Comparison of RNase A, a chemical cleavage and GC-clamped denaturing gradient gel electrophoresis for the detection of mutations in exon 9 of the human acid beta-glucosidase gene.

Gaucher disease (GD), which results from mutations in the human acid beta-glucosidase (beta-Glc) gene, was used as a model system to compare the utility of three methods capable of detecting single base substitutions. PCR-amplified beta-Glc exon 9 sequences of GD patients were screened for single base mutations by GC-clamped denaturing gradient gel electrophoresis (DGGE) and RNase A cleavage of RNA-DNA heteroduplexes, and by chemical (hydroxylamine/osmium tetroxide) cleavage of dsDNA heteroduplexes. PCR products showing abnormal behaviour were cloned and sequenced. Three new point mutations were detected by this strategy. A G to C (Asp409 to His409) substitution was present in two Type 1 and one Type 3 GD patients; an A to T transversion (Asp409 to Val409) was detected in only a single Type 3 individual, and a G to T mutation (Val394 to Leu394) was present in one Type 1 and one Type 3 patient. GD thus exhibits extensive molecular heterogeneity, with at least five single base mutations in beta-Glc exon 9. In every case verified by ASO hybridization, DGGE had correctly identified the presence of the three new mutations, as well as the two previously described exon 9 mutations. In comparison, although RNase A and the chemical method were both able to detect some of these mutations, neither method reproducibly detected all of them. Additionally, DGGE was the only method that was able to reliably determine whether a given mutation was present homozygously or heterozygously. These results suggest that GC-clamped DGGE may be a more reliable and informative screening method for point mutation detection.     

A method to detect ras point mutations in small subpopulations of cells.

Biochemical assays for ras mutations are capable of detecting a mutant allele only if it is present in at least 5% of cells tested. Further, ras mutation assays which utilize the polymerase chain reaction (PCR) are unable to distinguish a ras mutation in a small population of cells from mutations resulting from Taq DNA polymerase base misincorporation. We used a standard restriction fragment length polymorphism assay of PCR-amplified c-Ki-ras to detect codon 12 mutations in tumor cells and found a cumulative error frequency for Taq DNA polymerase of one codon 12 mutation per 2 X 10(4) molecules of total amplification product. The Taq polymerase-induced mutations were found to be multiple base transitions and represented a constant proportion of the amplification product at each step of the PCR. The ability to detect the in vitro generated mutation was dependent on the number of thermal cycles and the sensitivity of the detection assay. With these considerations in mind, we developed a two-step RFLP assay in which the thermal cycle number was kept low and molecules containing mutations at codon 12 were selectively amplified in the second step. We were able to detect a ras mutation occurring in 1 per 1000 cells (a two log improvement over standard RFLP methods) without detecting mutations resulting from Taq DNA polymerase infidelity.     

Identification of a beta-thalassemia mutation associated with a novel haplotype of RFLPs.

The study of the molecular defects that result in beta-thalassemia in Mediterraneans has uncovered a large number of unique mutations. This information is already being utilized for prenatal diagnosis of pregnancies at risk. Here, we report the definitive identification, by molecular cloning, of the beta-thalassemia mutation associated with a Mediterranean chromosome bearing a novel haplotype of restriction fragment length polymorphisms (RFLPs) in the beta gene cluster that has been previously designated as haplotype X. The thalassemia mutation was identified as a T----C base substitution at IVS-1 position 6, a mutation previously described in association with haplotype VI. We describe the use of the restriction enzyme SfaNI for the detection of this mutation and point out a possible pitfall that should be avoided if such an approach is used for the detection of this mutation, which appears to be a common cause of mild beta+-thalassemia in some populations.     

Detection of rare point mutation via allele-specific amplification in emulsion PCR

It is essential to analyze rare mutations in many fields of biomedical research. However, the detection of rare mutations is usually failed due to the interference of predominant wild-type DNA surrounded. Herein we describe a sensitive and facile method of detecting rare point mutation on the basis of allele-specific amplification in emulsion PCR. The identification and selective amplification of rare mutation are accomplished in one-pot reaction. The allele-specific primers coupled on magnetic beads allow the exclusive amplification and enrichment of the mutant amplicons. The productive beads bearing mutant amplicons are subsequently stained with the fluorescent dyes. Thus, the rare point mutations with a percentage as low as 0.1%, can be detected by fluorescent analysis. The relative percentages of mutation among different samples can be roughly accessed by counting the fraction of fluorescent positive beads through flow cytometry. [BMB Reports 2013; 46(5): 270-275]     

An isothermal method for whole genome amplification of fresh and degraded DNA for comparative genomic hybridization, genotyping and mutation detection.

Molecular genotyping has important biomedical and forensic applications. However, limiting amounts of human biological material often yield genomic DNA (gDNA) in insufficient quantity and of poor quality for a reliable analysis. This motivated the development of an efficient whole genome amplification method with quantitatively unbiased representation usable on fresh and degraded gDNA. Amplification of fresh frozen, formalin-fixed paraffin-embedded (FFPE) and DNase-degraded DNA using degenerate oligonucleotide-primed PCR or primer extension amplification using a short primer sequence bioinformatically optimized for coverage of the human genome was compared with amplification using current primers by chromosome-based and BAC-array comparative genomic hybridization (CGH), genotyping at short tandem repeats (STRs) and single base mutation detection. Compared with current primers, genome amplification using the bioinformatically optimized primer was significantly less biased on CGH in self-self hybridizations, and replicated tumour genome copy number aberrations, even from FFPE tissue. STR genotyping could be performed on degraded gDNA amplified using our technique but failed with multiple displacement amplification. Of the 18 different single base mutations 16 (89.5%) were correctly identified by sequencing gDNA amplified from clinical samples using our technique. This simple and efficient isothermal method should be helpful for genetic research and clinical and forensic applications.     

四引物PCR扩增反应的单管SNP快速测定法

建立一种在单管中进行单核苷酸多型性 (SNP)快速测定的高效廉价方法 .以人ABCA1基因中的I82 3M为研究对象 ,设计 4种引物进行PCR扩增 ,其中两种引物用于扩增一段含有SNP位点的DNA片段 ,另两种引物为SNP位点特异性引物 ,4种引物在单管中同时进行PCR扩增反应 ,根据延伸产物的长度确定SNP的类型 .为提高SNP测定的特异性 ,在特异性引物的 3′端倒数第 3个碱基引入了一个人为错配碱基 ,使引物的错误延伸率显著降低 ,大大提高了SNP分析的准确性 .实验结果表明 ,所建立的方法简单 ,操作简便 ,可在单管中完成SNP的测定反应 .     

The detection of beta-globin gene mutations in beta-thalassemia using oligonucleotide probes and amplified DNA

DNA amplification combined with the use of synthetic oligonucleotide probes has become an important tool in the identification of base substitutions. We report the use of this DNA amplification technique for the detection of mutations in beta-thalassemia. A series of oligonucleotide primers are synthesized which span the beta-globin gene; one primer is complementary to the coding strand and the other to the non-coding strand. The primers are chosen so that there is little homology with other DNA segments, especially the delta gene. Each set of primers spans an area of the gene between 100 and 300 bp, while the suspected mutation point is located between these two primers. With the use of such a primer set, the beta-globin gene region is amplified by denaturation, annealing and DNA synthesis. The amplification cycle is repeated 25-30 times, using the Klenow fragment of DNA polymerase I. The resulting amplified DNA is hybridized with normal and synthetic deoxynucleotide probes using a standard dot-blot method. We have designed a set of primers and experimental conditions which should prove useful to diagnostic centers for detection of numerous beta-thalassemia mutations.     

Detecting single base substitutions as heteroduplex polymorphisms.

We have developed a sensitive technique for detecting single base substitutions in polymerase chain reaction (PCR) products from individuals heterozygous for polymorphisms or new mutations. This technique takes advantage of the formation of heteroduplexes in the PCR between different alleles from heterozygous individuals. These heteroduplexes can be detected on polyacrylamide gels because they migrate slower than their corresponding homoduplexes. Using PCR, we have generated a series of point mutations in a defined region of DNA in the equine infectious anemia virus (EIAV). Each mutation is the result of a single base substitution. By mixing the PCR products amplified from these mutations with one another, as well as with wildtype PCR products, we can generate heteroduplexes in which the identity of the mismatched bases is known. We detected eight of nine point mutations using this technique. We have also modified the electrophoretic conditions to optimize the detection of these heteroduplexes. In addition, the usefulness of this technique is demonstrated by its ability to detect a mutation in the cystic fibrosis gene that is the result of a single base substitution. This technique should prove useful for rapidly screening large numbers of individuals for new mutations or polymorphisms.     

A Rapid and Cost-Effective Method for Genotyping Genome-Edited Animals:A Heteroduplex Mobility Assay Using Microfluidic Capillary Electrophoresis

The recent emergence and application of engineered endonucleases have led to the development of genome editing tools capable of rapidly implementing various targeted genome editions in a wide range of species.Moreover,these novel tools have become easier to use and have resulted in a great increase of applications.Whilst gene knockout(KO) or knockin(KI) animal models are relatively easy to achieve,there is a bottleneck in the detection and analysis of these mutations.Although several methods exist to detect these targeted mutations,we developed a heteroduplex mobility assay on an automated microfluidic capillary electrophoresis system named HMA-CE in order to accelerate the genotyping process.The HMA-CE method uses a simple PCR amplification of genomic DNA(gDNA) followed by an automated capillary electrophoresis step which reveals a heteroduplexes(HD) signature for each mutation.This allows efficient discrimination of wild-type and genome-edited animals down to the single base pair level.     

A novel assay for allelic discrimination that combines the fluorogenic 5' nuclease polymerase chain reaction (TaqMan) and mismatch amplification mutation assay

Recently much attention has been focused on single nucleotide polymorphisms (SNPs) within fundamentally important genes, such as those involved in metabolism, cell growth regulation, and other disease-associated genes. Methodologies for discriminating different alleles need to be specific (robust detection of an altered sequence in the presence of wild-type DNA) and preferably, amenable to high throughput screening. We have combined the fluorogenic 5' nuclease polymerase chain reaction (TaqMan) and the mismatch amplification mutation assay (MAMA) to form a novel assay, TaqMAMA, that can quickly and specifically detect single base changes in genomic DNA. TaqMan chemistry utilizes fluorescence detection during PCR to precisely measure the starting template concentration, while the MAMA assay exploits mismatched bases between the PCR primers and the wild-type template to selectively amplify specific mutant or polymorphic sequences. By combining these assays, the amplification of the mutant DNA can be readily detected by fluorescence in a single PCR reaction in 2 hours. Using the human TK6 cell line and specific HPRT-mutant clones as a model system, we have optimized the TaqMAMA technique to discriminate between mutant and wild-type DNA. Here we demonstrate that appropriately designed MAMA primer pairs preferentially amplify mutant genomic DNA even in the presence of a 1,000-fold excess of wild-type DNA. The ability to selectively amplify DNAs with single nucleotide changes, or the specific amplification of a low copy number mutant DNA in a 1,000-fold excess of wild-type DNA, is certain to be a valuable technique for applications such as allelic discrimination, detection of single nucleotide polymorphisms or gene isoforms, and for assessing hotspot mutations in tumor-associated genes from biopsies contaminated with normal tissue.     

Needle-in-a-haystack detection and identification of base substitution mutations in human tissues

Background and induced germline mutagenesis and other genotoxicity studies have been hampered by the lack of a sufficiently sensitive technique for detecting mutations in a small cluster of cells or a single cell in a tissue sample composed of millions of cells. The most frequent type of genetic alteration is intragenic. The vast majority of oncogenic mutations in human and mammalian cancer involves only single base substitutions. We have developed universally applicable techniques that not only provide the necessary sensitivity and specificity for site specific mutagenesis studies, but also identify the point mutation. The exponential amplification procedures of polymerase chain reaction (PCR) and ligase chain reaction (LCR) have been combined with restriction endonuclease (RE) digestion to enable the selective enrichment and detection of single base substitution mutations in human oncogenic loci at a sensitivity of one mutant in more than 10⁷ wild type alleles. These PCR/RE/LCR procedures have been successfully designed and used for codons 12 and 248 of the Ha-ras and p53 genes, respectively, both of which contain a natural MspI restriction endonuclease recognition sequence. These procedures have also been adapted for the detection and identification of mutations in oncogenic loci that do not contain a natural restriction endonuclease recognition sequence. Using PCR techniques, a HphI site was incorporated into the codons 12/13 region of the human N-ras gene, which was then used for the selective enrichment of mutants at this oncogenic locus. These PCR/RE/LCR procedures for base substitution mutations in codon 12 of the N-ras gene were found to have the sensitivity of detection of at least one mutant allele in the presence of the DNA equivalent of 10⁶ wild type cells. Only one peripheral blood leukocyte DNA specimen out of nine normal individuals displayed an observable Ha-ras mutation that was present at frequency between 10⁻⁵ and 10⁻⁶. These PCR/RE/LCR techniques for detecting and identifying base substitution mutations are universally applicable to almost any locus or base site within the human or animal genome. With the added advantage of the adjustability of both the amount of DNA (number of genomes) to be tested and the sensitivity (10⁻² to 10⁻⁷) of the assay selection or enrichment procedures, these PCR/RE/LCR techniques will be useful in addressing a broad range of important questions in mutagenesis and carcinogenesis.     

Ligase-based detection of mononucleotide repeat sequences

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.