Showing papers in "BioTechniques in 1995"

Short technical reports. Modification of the TRI reagent procedure for isolation of RNA from polysaccharide- and proteoglycan-rich sources.

Abstract: A modification of the TRI Reagent procedure has been elaborated for isolation of RNA from polysaccharide- and proteoglycan-rich material. In the modified procedure, RNA is precipitated from the aqueous phase by the combined action of isopropanol and a high-salt concentration. Under these conditions, RNA is effectively precipitated while contaminating polysaccharides and proteoglycans remain in the soluble form. The modified precipitation does not prolong or increase the complexity of the TRI Reagent procedure. The new procedure was tested by isolation of RNA from polysaccharide- and proteoglycan-rich tissues such as rat liver and aorta.

Extraction, PCR amplification and sequencing of mitochondrial DNA from human hair shafts.

Abstract: Techniques have been developed for extracting, amplifying and directly sequencing mitochondrial DNA (mtDNA) from human hair shafts. The hair shaft is ground in a glass micro-tissue grinder, and the DNA is extracted with organic solvent and purified by filtration. The filtrate subsequently provides the mtDNA template for the PCR. The two hypervariable segments of the mtDNA control region are amplified in four separate reactions. After a purification step to remove unincorporated PCR primers, amplified products are quantitated by capillary electrophoresis and subjected to cycle sequencing. The products are separated and analyzed on an automated DNA sequencer. The mtDNA sequences from the hair shaft match the mtDNA sequences from blood samples taken from the same donor.

A pre-loading method of evaluating gap junctional communication by fluorescent dye transfer.

Abstract: We describe a simple method for evaluating gap junctional communication (GJC) between cells in culture. The procedure involves pre-loading cells with two fluorescent dyes: calcein and DiI. Calcein is able to pass through gap junctions, while DiI is not. These pre-loaded cells are then plated with unlabeled cells. The number of cells receiving calcein from each pre-loaded cell can then be quantified after the cells settle on the plate. Potent and reversible inhibitors of GJC can be used in this system to evaluate dye transfer within a given period of time.

Green fluorescent protein as a reporter of gene expression and protein localization.

Abstract: The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is rapidly becoming an important reporter molecule for monitoring gene expression and protein localization in vivo, in situ and in real time. GFP emits bright green light (lambda max = 509 nm) when excited with UV or blue light (lambda max = 395 nm, minor peak at 470 nm). The fluorescence excitation and emission spectra of GFP are similar to those of fluorescein, and the conditions used to visualize this fluorophore are also suitable for GFP. Unlike other bioluminescent reporters, the chromophore in GFP is intrinsic to the primary structure of the protein, and GFP fluorescence does not require a substrate or cofactor. GFP fluorescence is stable, species-independent and can be monitored non-invasively in living cells and, in the case of transparent organisms, whole animals. Here we demonstrate GFP fluorescence in bacterial and mammalian cells and introduce our Living Colors line of GFP reporter vectors, GFP protein and anti-GFP antiserum. The reporter vectors for GFP include a promoterless GFP vector for monitoring the expression of cloned promoters/enhancers in mammalian cells and a series of six vectors for creating fusion protein to either the N or C terminus of GFP.

Triple-label hybridization assay for type-1 diabetes-related HLA alleles.

Abstract: We describe a method for the detection of two type 1 (insulin-dependent) diabetes susceptibility ( * 0201, * 0302) alleles and two protective ( * 0301, * 0602/0603) alleles of the HLA-DQB1 gene on the human major histocompatibility complex (MHC). The test is based on DNA amplification with PCR followed by simultaneous, allele-specific triple-label hybridization performed in microtitration wells. In the hybridization, very short allele-specific oligonucleotides labeled with europium (Eu), terbium (Tb) or samarium (Sm) are used. The labeled probes could be detected using time-resolved fluorometry with sensitivities of 1×10 7 , 3×10 8 and 3×10 8 molecules, respectively. Cross-reactions were not found among samples containing 14 common DQB1 alleles. To test the utility of the developed assay, 100 DNA and 14 dried blood spot samples with known DQB1 alleles were analyzed. A 100% agreement with the reference method was reached. Thus, this triple-label hybridization assay proved to be suitable even for detection of a large number of samples

Single-step protocol for preparation of plant tissue for analysis by PCR.

Abstract: PCR has many applications in the isolation and analysis of plant DNA. The influence of salt and EDTA concentration, pH, incubation time and temperature on the preparation of plant material for PCR was evaluated. A general single-step method was developed in which a small amount of plant tissue was heated in a simple solution. The DNA in the supernatant was found to be suitable for most PCR applications including arbitrarily primed PCR (random-amplified polymorphic DNA) and PCR with specific primers for both single- and multiple-copy genes. The technique is much simpler than those generally used for plant DNA preparation and was successful with tissues from a wide range of species.

Lipofection reagents prepared by a simple ethanol injection technique.

Abstract: Cationic liposomes are being utilized for the delivery of DNA into mammalian cells both in vitro and in vivo. In this report, we describe a rapid, simple injection method for the preparation of cationic liposomes that requires no special equipment. We demonstrate that, using this injection method, liposomes prepared with a commercially available cationic lipid (dimethyldioctadecylammonium bromide, DDAB) or a synthetic cationic cholesterol derivative, along with a neutral lipid (dioleoyl-L-alpha-phosphatidylethanolamine, DOPE), effectively transfect a variety of cell lines in vitro. The transfection activity of liposomes prepared by this method was comparable to that obtained with liposomes prepared by the standard evaporation/sonication procedure and to that of a commercial reagent, Lipofectamine.

New primer strategy improves precision of differential display

Abstract: To increase the reproducibility and to reduce the false positives in the initial mRNA differential display, modified long composite primers were developed based on both mRNA differential display and RNA arbitrarily primed PCR fingerprinting methods. Ten-base nucleotides were added at the 5' ends of the primers used in the initial mRNA differential display. These included a restriction site to aid cloning. PCR began with one low-stringency cycle (40 degrees C for annealing) followed by 35 high-stringency cycles (60 degrees C for annealing). The modified method significantly improved the reproducibility and sensitivity of the mRNA differential display while still keeping the characteristics of the original method.

In situ hybridization with digoxigenin-labeled RNA probes: facts and artifacts.

Abstract: We have developed an easy, stream-lined yet sensitive protocol for in situ hybridization to mRNA in frozen tissue sections or cytospins using digoxigenin-labeled RNA probes detected by alkaline phosphatase. We found the crucial parameters for successfully performing this technique to be tissue quality, fixation time and effective removal of excess probe. Most preparation, incubation steps and washes as described in the literature were found to be unnecessary and, therefore, eliminated, making this protocol simple enough to be achieved in any laboratory with access to tissue preparation and some molecular biology expertise.

Restriction endonuclease fingerprinting (REF): a sensitive method for screening mutations in long, contiguous segments of DNA.

Abstract: Restriction endonuclease fingerprinting (REF) is a modification of single-strand confirmation polymorphism (SSCP) that was developed to detect the presence of essentially all mutations in a 1-kb segment. To test REF, a 1-kb segment of the human factor IX gene was amplified with PCR and digested with each of five groups of restriction endonucleases. The endonucleases in each group were chosen so that the average size of the fragments was about 150 bp. After separate digestions, the products were mixed, 5' end-labeled with T4 polynucleotide kinase, denatured and electrophoresed under nondenaturing conditions. Each lane screened 1 kb and typically contained 68 segments (6.8 fragments per average digestion x 5 digestions x 2 strands). REF was performed with 5.6% polyacrylamide and 7.5% GeneAmp at temperatures of either 23 degrees or 8 degrees C. Point mutations resulted in the gain or loss of a restriction site in 21% of 24 test mutations (informative restriction component). In cases in which the restriction component was not informative, mutations were detected if any of the five mutation-containing restriction fragments (producing 10 single-stranded segments) displayed abnormal mobility (SSCP component). The average efficiency per single-stranded segment of the SSCP component for the 24 point mutations ranged from 49% for polyacrylamide at 23 degrees C to 68% with GeneAmp at 8 degrees C. REF detected 96% of the mutations with polyacrylamide at 23 degrees C and 100% with GeneAmp at 23 degrees or 8 degrees C. GeneAmp at 23 degrees and 8 degrees C also detected 100% of a subsequent blinded sample that contained normal controls and 27 different point mutations.(ABSTRACT TRUNCATED AT 250 WORDS)

Highly discriminating heptaplex short tandem repeat PCR system for forensic identification.

Abstract: We describe a highly discriminating multiplex short tandem repeat PCR human identification system that gives a matching probability for Caucasians of European ancestry of 2.94 x 10(-8) or 5.66 x 10(-10) when used in combination with a previously described system. The system produces discrimination equal to or greater than four single locus probes (restriction fragment length polymorphism [RFLP] typing of variable nucleotide tandem repeat [VNTR] loci). The test is robust and reproducible and works with 1-10 ng of template DNA, using fluorescent detection of PCR products from either 4 or 6 short tandem repeat loci and the X-Y homologous gene amelogenin, giving simultaneous sex diagnosis.

Quantitative RT-PCR for measuring gene expression.

Abstract: Classical Northern blot analysis for measuring mRNA requires too many cells to be practical for cell sorting. Yet, measurement of gene expression in small subsets within a heterogeneous population of cells is often desired. The PCR in combination with prior reverse transcription (RT-PCR) of the mRNA of interest provides a means for measuring gene expression using as few as one cell. When RT-PCR is performed, the reliability of the data can be highly subjective due to the efficiency of both RT and PCR steps. This subjectivity can be eliminated by a technique for quantitating specific RNA molecules using an internal RNA competitive reference standard (RNA-CRS), which is identical to the sequence of interest except for a deletion of 80 bases. Here we illustrate a strategy for quantitative PCR using a RNA-CRS, synthesized solely using nonplasmid-based PCR techniques. The competitive reaction consists of a constant quantity of wild-type mRNA (from 100-1000 cells) added individually to tubes containing a serially decreasing amount of RNA-CRS. The RT-PCR is performed on these samples, then the resulting product is analyzed by gel electrophoresis and densitometry. The procedure for preparing the RNA-CRS and subsequent RT-PCR steps are described in detail.

Genetic transformation of nematodes using arrays of micromechanical piercing structures

Abstract: Foreign genes were expressed in the nematode Heterorhabditis bacteriophora using a new micro-mechanical device to inject DNA-coated microprobe through the nematode cuticle. After introduction of a plasmid in which a Caenorhabditis elegans 16-kDa heat shock promoter was fused to Escherichia coli, a beta-galactosidase gene was expressed in the progeny of the injected worms. The tip of the microprobe penetrated through the nematode cuticle without causing injury to the nematode, and about 8% of the total progeny tested expressed the foreign gene. We describe and validate a new and efficient genetic transformation system for nematodes. The method is quick and easy to use.

Detection of wild-type contamination in a recombinant adenoviral preparation by PCR

Abstract: A rapid and sensitive method of detecting wild-type virus contamination is needed for the preparation of recombinant adenoviruses for adenoviral vector applications for adenoviral vector applications in which purified vectors free of wild-type virus are required for preclinical studies and clinical trials. In response to this demand, we developed a PCR assay that uses two pairs of primers in the same reaction to detect adenoviral El DNA with co-amplification of E2B DNA as an internal control. Template DNA preparation was simplified and required only 365 μL of culture medium of 293 cells that displayed a cytopathic effect following adnovirus infection. Evaluation of the sensitivity of the assay demonstrated that it detected the El DNA in a reconstitution of one plaque-forming unit (pfu) of wild-type virus in 10 9 pfu of recombinant viruses. This method may be useful for quality control in the production of adenoviral vectors free of wild-type virus for gene therapy applications

Short tandem repeat typing of bodies from a mass disaster: high success rate and characteristic amplification patterns in highly degraded samples.

Abstract: We have used a PCR-based DNA-typing method, involving the coamplification of four tetrameric short tandem repeat loci, in the analysis of a large number of severely degraded tissue samples taken from the scene of a mass disaster in which bodies were exposed to extreme thermal, physical and chemical insult. Analysis of the amplified DNA in a number of the samples revealed uniquely sized artifact PCR products resulting from the amplification of degraded genomic DNA as well as characteristic patterns in the amounts of PCR products generated from differently sized loci. This system has proved to be very reliable and robust, and we were successful in typing all of the four loci in 66% of the samples tested and at least one locus in 83% of the cases. A PCR-based sex test also proved to be very effective when applied to the degraded samples.

Simultaneous detection of TDT-mediated dUTP-biotin nick end-labeling (TUNEL)-positive cells and multiple immunohistochemical markers in single tissue sections.

Abstract: We have modified the terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-biotin nick end-labeling (TUNEL) method to permit the immunogold-silver intensification detection of apoptotic cells in tissue sections. Such sections can subsequently be processed for multi-labeling fluorescence microscopy, thus permitting the simultaneous detection of silver-positive apoptotic cells and three additional fluorescent signals. In this report, we combined TUNEL labeling with the fluorescent detection of bromodeoxyuridine-positive proliferating cells, growth-associated protein-43 (GAP-43) immunoreactive neurons and bisbenzimide-positive pyknotic cells in the embryonic mouse nervous system. This combination of stains allowed the simultaneous detection of proliferative, differentiated and apoptotic cells in the developing nervous system.

Sequence-specific purification of nucleic acids by PNA-controlled hybrid selection

Abstract: Using an oligohistidine peptide nucleic acids (oligohistidine-PNA) chimera, we have developed a rapid hybrid selection method that allows efficient, sequence-specific purification of target nucleic acid. The method exploits two fundamental features of PNA. First, that PNA binds with high affinity and specificity to its complementary nucleic acid. Second, that amino acids are easily attached to the PNA oligomer during synthesis. We show that a (His) 6 -PNA chimera exhibits strong binding to chelated Ni 2+ ions without compromising its native PNA hybridization properties. We further show that these characteristics allow the (His) 6 -PNA/DNA complex to be purified by the well-established method of metal ion affinity chromatography using a Ni 2+ -NTA (nitrilotriactic acid) resin. Specificity and efficiency are the touch-stones of any nucleic acid purification scheme. We show that the specificity of the (His) 6 -PNA selection approach is such that oligonucleotides differing by only a single nucleotide can be selectively purified. We also show that large RNAs (2224 nucleotides) can be captured with high efficiency by using multiple (His) 6 -PNA probes. PNA can hybridize to nucleic acids in low-salt concentrations that destabilize native nucleic acid structures. We demonstrate that this property of PNA can be utilized to purify an oligonucleotide in which the target sequence forms part of an intramolecular stem/loop structure.

Secondary structure in the 3' UTR of EGF and the choice of reverse transcriptases affect the detection of message diversity by RT-PCR.

Abstract: The secondary structure in mRNA is essential for many processes, but it can present a technical problem in making full-length cDNA with reverse transcriptases. Furthermore, different reverse transcriptases have differing abilities to transcribe through regions with secondary structure, which can alter the products obtained by reverse-transcribing RNA and then PCR-amplifying the product (RT-PCR). We have been interested in studying the posttranscriptional regulation of epidermal growth factor by RT-PCR and have tested the ability of several reverse transcriptases to reverse transcribe the 3'-untranslated region (3'UTR), a region that contains substantial secondary structure. When low levels of either total RNA or poly(A)+ mRNA were used, we found avian myeloblastosis virus reverse transcriptase (AMV-RT) to be the most robust of all the enzymes tested. Furthermore, contrary to reports that AMV-RT is inhibited by tRNA--which should make it less effective than Moloney murine leukemia virus reverse transcriptase (MMLV-RT) at reverse-transcribing total RNA--adding tRNA to poly(A)+ RNA actually increased the amount of specific RT-PCR product obtained with AMV-RT while it decreased the amount of product and enhanced mispriming with MMLV-RT. We found that pre-incubation of the oligo(dT) primer with total RNA at elevated temperature prior to reverse transcription improved the efficiency of both native and modified MMLV-RTs. These findings support the concept that secondary structures in RNA differentially affect the abilities of different reverse transcriptases to detect transcript diversity and raise the possibility that such structures could affect quantitation using RT-PCR with internal mRNA standards.

Single-step purifications of His6-MutH, His6-MutL and His6-MutS repair proteins of escherichia coli K-12.

Abstract: The MutS, MutH and MutL proteins mediate methyl-directed-mismatch (MDM) repair in Escherichia coli and Salmonella typhimurium. These proteins have been developed into powerful tools for screening genomes for polymorphisms and detecting and localizing mutations. In an ongoing study of the regulation of MDM repair, we developed one-step schemes to purify the E. coli MutS, MutH and MutL proteins fused to a polyhistidine (His6) affinity tag. The E. coli K-12 mutS+, mutH+ and mutL+ genes were cloned from the Clarke-Carbon plasmid or Kohara-phage library into expression vector pET-15b, which allows fusion to the His6 affinity tag. Each of the resulting recombinant plasmids complemented the corresponding mutHLS mutation in the Cupples-Miller CC106 mutator tester strain, indicating that the His6-MutHLS fusion proteins were individually functional in vivo. The His6-MutHLS proteins were separately purified by variations of batch binding to Ni(2+)-chelation affinity resin. The yield of purified His6-MutHLS proteins from these procedures was 0.4-0.6 mg from 40 mL of induced culture. The binding properties of one-step-purified His6-MutS protein were characterized further. His6-MutS exhibited the same mismatch-binding activity and specificity as native MutS in side-by-side bandshift assays. These constructs and purification methods should be useful to laboratories wishing to apply or develop MutS-mismatch mapping and to other applications or studies of the E. coli MutHLS repair proteins.