Identification of individuals by analysis of biallelic DNA markers, using PCR and solid-phase minisequencing.

We have developed a new method for forensic identification of individuals, in which a panel of biallelic DNA markers are amplified by the PCR, and the variable nucleotides are detected in the amplified DNA fragments by the solid-phase minisequencing method. A panel of 12 common polymorphic nucleotides located on different chromosomes with reported allele frequencies close to .5 were chosen for the test. The allele frequencies for most of the markers were found to be similar in the Finnish and other Caucasian populations. We also introduce a novel approach for rapid determination of the population frequencies of biallelic markers. By this approach we were able to determine the allele frequencies of the markers in the Finnish population, by quantitative analysis of three pooled DNA samples representing 3,000 individuals. The power of discrimination and exclusion of the solid-phase minisequencing typing test with 12 markers was similar to that of three VNTR markers that are routinely used in forensic analyses at our institute. The solid-phase minisequencing method was successfully applied to type paternity and forensic case samples. We also show that the quantitative nature of our method allows typing of mixed samples.     

Direct amplification of single-stranded DNA for pyrosequencing using linear-after-the-exponential (LATE)-PCR

Pyrosequencing is a highly effective method for quantitatively genotyping short genetic sequences, but it currently is hampered by a labor-intensive sample preparation process designed to isolate single-stranded DNA from double-stranded products generated by conventional PCR. Here linear-after-the-exponential (LATE)-PCR is introduced as an efficient and potentially automatable method of directly amplifying single-stranded DNA for pyrosequencing, thereby eliminating the need for solid-phase sample preparation and reducing the risk of laboratory contamination. These improvements are illustrated for single-nucleotide polymorphism genotyping applications, including an integrated single-cell-through-sequencing assay to detect a mutation at the globin IVS 110 site that frequently is responsible for beta-thalassemia.     

Effect of salicylic acid,methyl jasmonate,ethephon and cantharidin on anthraquinone production by Rubia cordifolia callus cultures transformed with the rolB and rolC genes

A novel method for immobilizing large DNA fragments on a solid surface was developed. A mixed self-assembled monolayer of thiolated single-stranded DNA with inert alkanethiol was generated on a gold (Au) surface through the Au-S reaction. Surface-tethered DNA generated by this method was compatible with various genetic engineering techniques, including hybridization, polymerization, restriction enzyme digestion and ligation. Kinetic control of surface coverage of immobilized DNA was critical for optimizing genetic engineering techniques on solid-phase. Multi-step reaction schemes utilizing various genetic engineering techniques described above were employed for solid-phase gene assembly. We were able to immobilize DNA fragments of up to 1180 bp on a solid surface. Furthermore, we showed that these immobilized genes can be regenerated by PCR. The present work suggests that these types of assembled genes can be used to store and regenerate genes on solid-phase.     

Analysis of polymerase chain reaction products by high-performance liquid chromatography with fluorimetric detection and its application to DNA diagnosis

We describe the development of a sensitive high-performance liquid chromatographic (HPLC) method for polymerase chain reaction (PCR) products using bisbenzimide (Hoechst 33258 dye) based fluorimetric detection. The detection limit and specificity for double-strand DNA detection are improved in comparison with HPLC with UV absorbance detection. This HPLC, using a column packed with diethylaminoethyl-bonded non-porous resin particles, was applied to the detection of allele-specific PCR and restriction fragment length polymorphism analysis. We also developed a hybridization method analyzed by HPLC. DNA fragments (149 bp) containing the mutation site (C→A,G,T) in the N-ras gene were amplified by PCR. Fluorescein isothiocyanate (FITC)-labeled DNA probes were also prepared by PCR using FITC-labeled 5′ primer. Analysis of mutation was performed by the separation of a hybrid and non-reactive DNA probe with HPLC with fluorimetric detection after the hybridization of target DNA (149 bp) and a FITC DNA probe. The effects of various factors on hybridization were examined to establish optimal assay conditions. Under the conditions determined, a point mutation in PCR products obtained from the N-ras gene could be detected specifically by this method. The analysis of PCR products by HPLC may potentially be useful for DNA diagnosis.     

寡核苷酸DNA Microarray用于HLA DRB1基因分型的研究

对寡核苷酸DNA Microarray用于HLA DRB1基因分型的技术进行研究。常规的酚/氯仿法提取标准血样基因组DNA,在DRB1的exon2区域设计一对引物,经PCR扩增基因组相应区段并用Cy5-dCTP进行标记。设计寡核苷酸分型探针,将探针固定在APS-PDC法制作的DNA Microarray上,用标记的PCR产物与之杂交,扫描仪对杂交效果进行扫描,Imagene软件对杂交图像进行分析。共检测了33例标准血样的HLA DRB1基因型。检测结果证明研制的DNA Microarray准确、灵敏。DNA Microarray技术可以有效地检测DRB1等位基因,对比常规的PCR-SSP和PCR-SSO方法、分型基因芯片方法更为直观,并有集成化优势。     

One-step isolation of plant DNA suitable for PCR amplification

We report a one-step extraction technique for the isolation of plant DNA, DNA suitable for amplification by PCR can be produced from leaf material smaller than 0.3 mm² in less than 20 min, with no tube changes. The method was tested on several plant specA00AK020ies. The described method was found to extract DNA that could be amplified without any further purification or treatment. The isolated DNA was amplified using a universal chloroplast primer set. The method was validated by comparing size of PCR products generated by the novel method to PCR products generated using standard DNA isolation techniques.     

Mutations in the gene encoding 21-hydroxylase detected by solid-phase minisequencing

We have developed an assay based on solid-phase minisequencing to screen for the following seven point mutations in the gene CYP21 encoding 21-hydroxylase: Pro30Leu, I2-splice, Ile172Asn, Cluster-E6, Val281Leu, Gln318Stop, and Arg356Trp. 5′-Biotinylated PCR products of CYP21 are bound to streptavidin-coated microtiter wells, where the minisequencing reaction takes place after denaturation of DNA. Depending on the sequence investigated, one specific ³H-labelled deoxyribonucleotide is incorporated to extend a detection primer. By using an appropriate set of detection primers, it is possible to screen the gene for several mutations within the same PCR amplificate. This fast and reliable method very clearly distinguishes between DNA from homozygous mutant, heterozygous, and normal individuals and is well suited for routine diagnosis of patients with 21-hydroxylase deficiency and for carrier detection. Received: 19 August 1996     

Cloning and direct sequencing of plant promoters using primer-adapter mediated PCR on DNA coupled to a magnetic solid phase.

A method that allows amplification and direct sequencing or cloning of an unknown DNA segment flanked by a known sequence is described using barley genomic DNA. The method avoids the step of circularization necessary for inverse PCR by ligation of primer-adapters to restricted genomic DNA. Specificity is achieved in the first amplification step; linear PCR with a biotinylated primer complementary to the known flanking sequence (primer 1-B) produces a single-stranded product that is purified employing streptavidin-coated magnetic beads. After this step, which removes genomic DNA, two rounds of exponential PCR are performed, first with the adapter-primer and primer 1 and second with primer 1 substituted by a nested primer 2. If the second primer is biotinylated, the product can be sequenced directly using solid-phase sequencing. We have employed this method to sequence directly and to clone the promoters of two late embryogenesis-abundant (Lea) genes (B19.4 and B19.3) from barley. Lea B19.4 and B19.3 encode putative desiccation-protective proteins that act in the final stages of embryogenesis and have previously been cloned as cDNAs. We demonstrate here that their proximal promoter regions are very similar (80% identity) and that both contain putative abscisic acid-responsive elements.     

Solid-phase cloning for high-throughput assembly of single and multiple DNA parts

We describe solid-phase cloning (SPC) for high-throughput assembly of expression plasmids. Our method allows PCR products to be put directly into a liquid handler for capture and purification using paramagnetic streptavidin beads and conversion into constructs by subsequent cloning reactions. We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of >60 000 unique human gene fragments into expression vectors. In addition, we report on SPC-based single-strand assembly for applications where exact control of the sequence between fragments is needed or where multiple inserts are to be assembled. In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in. The usefulness of head-to-tail SPC was demonstrated by assembly of >150 constructs with up to four DNA parts at an average success rate above 80%. We report on several applications for SPC and we suggest it to be particularly suitable for high-throughput efforts using laboratory workstations.     

Removal of PCR error products and unincorporated primers by metal-chelate affinity chromatography

Immobilized Metal Affinity Chromatography (IMAC) has been used for decades to purify proteins on the basis of amino acid content, especially surface-exposed histidines and "histidine tags" genetically added to recombinant proteins. We and others have extended the use of IMAC to purification of nucleic acids via interactions with the nucleotide bases, especially purines, of single-stranded RNA and DNA. We also have demonstrated the purification of plasmid DNA from contaminating genomic DNA by IMAC capture of selectively-denatured genomic DNA. Here we describe an efficient method of purifying PCR products by specifically removing error products, excess primers, and unincorporated dNTPs from PCR product mixtures using flow-through metal-chelate affinity adsorption. By flowing a PCR product mixture through a Cu(2+)-iminodiacetic acid (IDA) agarose spin column, 94-99% of the dNTPs and nearly all the primers can be removed. Many of the error products commonly formed by Taq polymerase also are removed. Sequencing of the IMAC-processed PCR product gave base-calling accuracy comparable to that obtained with a commercial PCR product purification method. The results show that IMAC matrices (specifically Cu(2+)-IDA agarose) can be used for the purification of PCR products. Due to the generality of the base-specific mechanism of adsorption, IMAC matrices may also be used in the purification of oligonucleotides, cDNA, mRNA and micro RNAs.     

DNA microarray probe preparation by gel isolation nested PCR

To develop a simplified method that can rapidly prepare DNA microarray probes in a massive scale, a lambda phage genomic DNA-fragments library was constructed for the microarray-probes collection. Four methods of DNA band recovery from the first PCR products were tested and compared. The DNA microarray probes were collected by a novel method of nested PCR that was mediated by gel isolation of the first PCR products. This method was named GIN-PCR. The probes that were prepared by this GIN-PCR technique were used as subjects to fabricate a DNA microarray. The results showed that a wooden toothpick was superior to the other 3 methods, since this technique can steadily transfer the DNA bands as the template of the second PCR after the first PCR. A group of probes were successfully collected and DNA microarrays were constructed using these probes. Hybridization results demonstrated that this technique of DNA recovery and probe preparation was rapid, efficient, and effective. We developed a cost-effective and less labor-intensive method for DNA microarray probe preparation by nested PCR that is mediated by wooden toothpick transfer of the DNA bands in the gel after electrophoresis.     

Terminal transferase-dependent PCR: a versatile and sensitive method for in vivo footprinting and detection of DNA adducts.

We report here a new, sensitive and versatile genomic sequencing method, which can be used for in vivo footprinting and studies of DNA adducts. Starting with mammalian genomic DNA, single-stranded products are made by repeated primer extension; these products are subjected to homopolymeric ribonucleotide tailing at the 3' termini with terminal deoxynucleotidyl transferase and then ligated to a double-stranded linker having a complementary 3' overhang, and used for PCR. This terminal transferase-dependent PCR (TDPCR) method can generate band signals many-fold stronger than conventional ligation-mediated PCR (LMPCR). A UV photofootprint in the mouse Xist gene promoter can be easily detected using TDPCR. No special enzymes or chemical reagents are needed to convert DNA adducts into strand breaks. Any lesion that blocks primer extension should be detectable.     

Random PCR-based screening for soluble domains using green fluorescent protein

A novel approach to screen soluble protein domains is presented, by combining tagged random primer polymerase chain reaction (PCR) method and protein-folding assay using green fluorescent protein. Tagged random primer PCR method was used to amplify random DNA fragments from a template cDNA. The PCR products were fused to the green fluorescent protein (GFP) gene. Then solubilities of their translation products were rapidly monitored by the fluorescence of transformed Escherichia coli colonies on plates. We succeeded in cloning four soluble domains from Vav protein using this method. The present method is applicable to all proteins when cDNAs are available.     

Diagnosis of alpha 1-antitrypsin deficiency by enzymatic amplification of human genomic DNA and direct sequencing of polymerase chain reaction products.

We have compared sequencing of cloned "polymerase chain reaction" (PCR) products and the direct sequencing of PCR products in the examination of individuals from six families affected with alpha 1-antitrypsin (AAT) deficiency. In families where paternity was in question we confirmed consanguinity by DNA fingerprinting using a panel of locus-specific minisatellite probes. We demonstrate that direct sequencing of PCR amplification products is the method of choice for the absolutely specific diagnosis of AAT deficiency and can distinguish normals, heterozygotes and homozygotes in a single, rapid and facile assay. Furthermore, we demonstrate the reproducibility of the PCR and a rapid DNA isolation procedure. We have also shown that two loci can be simultaneously amplified and that the PCR product from each locus can be independently examined by direct DNA sequencing.     

PCR- and ligation-mediated synthesis of marker cassettes with long flanking homology regions for gene disruption in Saccharomyces cerevisiae.

We developed a novel method for synthesizing marker-disrupted alleles of yeast genes. The first step is PCR amplification of two sequences located upstream and downstream of the reading frame to be disrupted. Due to the addition of non-specific single A overhangs by Taq DNA polymerase, each PCR product can be ligated with a marker DNA which has T residues at its 3' ends. After amplification of individual ligation products through the second PCR, both products are mixed and annealed, and the single strand is converted to a double strand by an extension reaction. The final step is PCR amplification of the fragment composed of a selectable marker and two flanking sequences with the outermost primers. This method is rapid and needs only short oligonucleotides as primers.     

Detection of ligation products of DNA linkers with 5'-OH ends by denaturing PAGE silver stain

To explore if DNA linkers with 5'-hydroxyl (OH) ends could be joined by commercial T4 and E. coli DNA ligase, these linkers were synthesized by using the solid-phase phosphoramidite method and joined by using commercial T4 and E. coli DNA ligases. The ligation products were detected by using denaturing PAGE silver stain and PCR method. About 0.5-1% of linkers A-B and E-F, and 0.13-0.5% of linkers C-D could be joined by T4 DNA ligases. About 0.25-0.77% of linkers A-B and E-F, and 0.06-0.39% of linkers C-D could be joined by E. coli DNA ligases. A 1-base deletion (-G) and a 5-base deletion (-GGAGC) could be found at the ligation junctions of the linkers. But about 80% of the ligation products purified with a PCR product purification kit did not contain these base deletions, meaning that some linkers had been correctly joined by T4 and E. coli DNA ligases. In addition, about 0.025-0.1% of oligo 11 could be phosphorylated by commercial T4 DNA ligase. The phosphorylation products could be increased when the phosphorylation reaction was extended from 1 hr to 2 hrs. We speculated that perhaps the linkers with 5'-OH ends could be joined by T4 or E. coli DNA ligase in 2 different manners: (i) about 0.025-0.1% of linkers could be phosphorylated by commercial T4 DNA ligase, and then these phosphorylated linkers could be joined to the 3'-OH ends of other linkers; and (ii) the linkers could delete one or more nucleotide(s) at their 5'-ends and thereby generated some 5'-phosphate ends, and then these 5'-phosphate ends could be joined to the 3'-OH ends of other linkers at a low efficiency. Our findings may probably indicate that some DNA nicks with 5'-OH ends can be joined by commercial T4 or E. coli DNA ligase even in the absence of PNK.     

A novel method for producing partial restriction digestion of DNA fragments by PCR with 5-methyl-CTP.

Partial digestion of DNA fragments is a standard procedure for subcloning analysis and for generating restriction maps. We have developed a novel method to generate a partial digestion for any DNA fragment that can be amplified by PCR. The method involves the incorporation of 5-methyl-dCTP into the PCR product to protect most of the restriction sites. As a result, complete digestion of the modified PCR products with a 5-methyl-dCTP-sensitive enzyme will produce an array of restriction fragments equivalent to a partial restriction enzyme digestion reaction done on unmethylated PCR products. This method reduces the time and material needed to produce partially-digested DNA fragments by traditional methods. Furthermore, using fluorescein-labeled primers in the reaction, we were able to detect the fluorescein-labeled end fragments resulting from the enzyme digestion using a fluorimager or anti-fluorescein-AP antibody and thus determine the restriction maps.     

Accessing single nucleotide polymorphisms in genomic DNA by direct multiplex polymerase chain reaction amplification on oligonucleotide microarrays

This study introduces a DNA microarray-based genotyping system for accessing single nucleotide polymorphisms (SNPs) directly from a genomic DNA sample. The described one-step approach combines multiplex amplification and allele-specific solid-phase PCR into an on-chip reaction platform. The multiplex amplification of genomic DNA and the genotyping reaction are both performed directly on the microarray in a single reaction. Oligonucleotides that interrogate single nucleotide positions within multiple genomic regions of interest are covalently tethered to a glass chip, allowing quick analysis of reaction products by fluorescence scanning. Due to a fourfold SNP detection approach employing simultaneous probing of sense and antisense strand information, genotypes can be automatically assigned and validated using a simple computer algorithm. We used the described procedure for parallel genotyping of 10 different polymorphisms in a single reaction and successfully analyzed more than 100 human DNA samples. More than 99% of genotype data were in agreement with data obtained in control experiments with allele-specific oligonucleotide hybridization and capillary sequencing. Our results suggest that this approach might constitute a powerful tool for the analysis of genetic variation.     

High-Resolution Detection of PCR Products from a Microsatellite Marker Using a Nonradioisotopic Technique

We report a safe, rapid, and economical method for polymerase chain reaction (PCR)-based genotype analysis using a microsatellite marker specific for the human chromosome 18 locus, D18S53. This method does not involve radioisotopes and makes use of ethidium bromide fluorescence to detect PCR products. Our method enables direct analysis and easy detection of PCR products on nondenaturing polyacrylamide gels. The genotyping using this method can be scaled up to 100 samples at one time by adding a step of “double loading” of samples in a single sequencing size gel. We could resolve PCR products and DNA fragments, differing in size by only 2 bp, in the range of 150–200 bp by a 7% nondenaturing polyacrylamide gel. This technique can be applied for population-based genomic screening and linkage analysis.