Gene synthesis by circular assembly amplification

Here we report the development of a gene-synthesis technology, circular assembly amplification. In this approach, we first constructed exonuclease-resistant circular DNA via simultaneous ligation of oligonucleotides. Exonuclease- and subsequent mismatch cleaving endonuclease-mediated degradation of the resulting ligation mixture eliminated error-rich products, thereby substantially improving gene-synthesis quality. We used this method to construct genes encoding a small thermostable DNA polymerase, a highly repetitive DNA sequence and large (>4 kb) constructs.     

Use of a real-time polymerase chain reaction thermocycler to study bacterial cell permeabilization by antimicrobial peptides

Antimicrobial peptides are good leads to develop new antibiotics, but knowledge of their mode of action is a prerequisite. Destruction of the microbial membranes through a detergent-like mechanism is one of these modes of action. This is usually studied by using a fluorescent nucleic acid stain such as SYTOX Green, which is impermeable to living cells. Using a simple protocol based on the use of a standard real-time thermocycler, we confirmed that the actions of the antimicrobial peptides LL-37 and magainin 2 on bacterial cells are different.     

Rapid polymerase chain reaction amplification using intact bacterial cells

We have demonstrated that efficient polymerase chain reaction amplifications from chromosomal DNA can be carried out using whole bacterial cells as the starting material. Cells from the liquid or solid cultures can be used directly, without any pre-treatment, thus eliminating the need for DNA isolation.     

Gene walking using sequential hybrid primer polymerase chain reaction

We developed a simple and robust method for removing nonspecific amplification produced during gene walking with a gene-specific primer and a degenerate primer. The primary walking polymerase chain reaction (PCR) was followed by two or three PCR rounds, each incorporating a low concentration of a reverse hybrid primer, where the 3′ end was bound to a target sequence generated in the preceding PCR round and the 5′ end was a new sequence that generated a target sequence for the next PCR round. The low concentration of the hybrid primer and the extent of amplicon stem-loop formation inhibited nonspecific amplification and enabled successful walking along three genes.     

Differential amplification of rRNA genes by polymerase chain reaction.

The polymerase chain reaction (PCR) is used widely to recover rRNA genes from naturally occurring communities for analysis of population constituents. We have found that this method can result in differential amplification of different rRNA genes. In particular, rDNAs of extremely thermophilic archaebacteria often cannot be amplified by the usual PCR methods. The addition of 5% (wt/vol) acetamide to a PCR mixture containing both archaebacterial and yeast DNA templates minimized nonspecific annealing of the primers and prevented preferential amplification of the yeast small-subunit rRNA genes.     

Direct polymerase chain reaction (PCR) from human whole blood and filter-paper-dried blood by using a PCR buffer with a higher pH

We described a novel approach to directly amplify genomic DNA from whole blood and dried blood spotted on filter paper without any DNA isolation by using the PCR buffer with a higher pH, which was optimized as pH 9.1-9.6. Direct PCR on blood treated with various anticoagulants showed that the buffer worked well with the blood treated by citrate, EDTA, or heparinate. DNA fragments with different lengths could be efficiently amplified directly from various forms of blood samples. By coupling the buffer with tetra-PCR, a "true" single-tube genotyping was realized by using whole blood or paper-dried blood as starting material.     

Fingerprinting human chromosomes by polymerase chain reaction-mediated DNA amplification.

We describe here a method for DNA fingerprinting of human chromosomes by Alu-polymerase chain reaction (PCR) amplification of DNA from monochromosomal hybrids, following digestion with restriction endonucleases. DNA digestion with restriction enzymes prior to PCR amplification reduces the total number of amplified fragments. The number and pattern of bands of PCR products observed in an electrophoretic medium are chromosome specific and provide a "fingerprint signature" for individual human chromosomes. Using this approach, we have produced fingerprints for human chromosomes 2, 5, 7, 9, and 12. The applicability of this approach to chromosome identification was assessed by comparing the fingerprints obtained for two different hybrids containing chromosome 7. DNA fragments specific for the long and the short arms of human chromosome 12 have also been identified. In addition, Alu-PCR-generated DNA fragments, specific for different chromosomes, were used to probe Southern blots of a hybrid cell panel to identify human chromosomes present in hybrid cell lines. The chromosomal specificity of these probes permits the identification of intact as well as rearranged chromosomes composed of segments arising from more than one chromosome.     

Differential amplification of rRNA genes by polymerase chain reaction.

The polymerase chain reaction (PCR) is used widely to recover rRNA genes from naturally occurring communities for analysis of population constituents. We have found that this method can result in differential amplification of different rRNA genes. In particular, rDNAs of extremely thermophilic archaebacteria often cannot be amplified by the usual PCR methods. The addition of 5% (wt/vol) acetamide to a PCR mixture containing both archaebacterial and yeast DNA templates minimized nonspecific annealing of the primers and prevented preferential amplification of the yeast small-subunit rRNA genes.     

Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction

Gene Splicing by Overlap Extension or "gene SOEing" is a PCR-based method of recombining DNA sequences without reliance on restriction sites and of directly generating mutated DNA fragments in vitro. By modifying the sequences incorporated into the 5'-ends of the primers, any pair of polymerase chain reaction products can be made to share a common sequence at one end. Under polymerase chain reaction conditions, the common sequence allows strands from two different fragments to hybridize to one another, forming an overlap. Extension of this overlap by DNA polymerase yields a recombinant molecule. This powerful and technically simple approach offers many advantages over conventional approaches for manipulating gene sequences.     

Direct amplification of unknown genes and fragments by Uneven polymerase chain reaction

Gene cloning is a time-consuming task for molecular biologists, because it often takes weeks or months to construct, screen and finally clone a gene from a DNA library. Thus, more effective methods are needed for gene cloning. This paper describes a modified polymerase chain reaction (PCR) cycling condition, Uneven PCR, to generate specific unknown fragments or genes directly from total DNA instead of cloning fragments from DNA libraries. The essence of the method is to use two different annealing temperatures in consecutive PCR cycles to effectively amplify the target products while inhibiting the synthesis of non-specific products. Under favorable conditions, a desired DNA fragment or gene in the size range up to approximately 4 kb can be obtained and ready for cloning within a day or two.     

HLA-DR typing using DNA amplification by the polymerase chain reaction and sequential hybridization to sequence-specific oligonucleotide probes

A series of sequence-specific oligonucleotides (SSOs) have been used to type alleles at the HLA-DRB1 locus. Genomic DNA was amplified to high copy number by the polymerase chain reaction (PCR) and hybridizations of the dot-blotted, amplified DNA to a series of 14 SSOs enabled the identification of the major specificities DR1-DRw14. Certain alleles (DR3 and DR4) could be rapidly and accurately identified by running the products of allele-specific amplification of genomic DNA on agarose gels. This approach facilitated the typing of serological specificities such as subtypes of DR3 (DRw17 and DRw18) as well as alleles previously detected by the mixed lymphocyte reaction including subtypes of DR4 (Dw4, Dw10, Dw13, Dw14, and Dw15). The HLA-DR types obtained by SSO probing conformed to rules of Mendelian inheritance when they were applied to a series of 75 families. A full DR type could be obtained from many individuals simultaneously without needing to separate or store viable lymphocytes. Thus, this technique may have considerable implications for the analysis of disease associations with HLA class II alleles, particularly in circumstances where facilities for the initial preparation and storage of the samples may be limited.     

Sex identification of pigs using polymerase chain reaction amplification of the amelogenin gene

The amelogenin (AMEL) gene exists on both sex chromosomes of various mammalian species and the length and sequence of the noncoding regions differ between the two chromosome-specific alleles. Because both forms can be amplified using a single primer set, the use of AMEL in polymerase chain reaction (PCR)-based methods has facilitated sex identification in various mammalian species, including cattle, sheep and humans. In this study, we designed PCR primers to yield different-sized products from the AMEL genes on the X (AMELX) and Y (AMELY) chromosomes of pigs. PCR amplification of genomic DNA samples collected from various breeds of pigs (European breeds: Landrace, Large White, Duroc and Berkshire; Chinese breeds: Meishan and Jinhua and their crossbreeds) yielded the expected products. For all breeds, DNA from male pigs produced two bands (520 and 350 bp; AMELX and AMELY, respectively), whereas samples from female pigs generated only the 520 bp product. We then tested the use of PCR of AMEL for sex identification of in vitro-produced (IVP) porcine embryos sampled at 2 or 5 to 6 days after fertilization; germinal vesicle (GV)-stage oocytes and electroactivated embryos were used as controls. More than 88% of the GV-stage oocytes and electroactivated embryos yielded a single 520 bp single band and about 50% of the IVP embryos tested produced both bands. Our findings show that PCR analysis of the AMEL gene is reliable for sex identification of pigs and porcine embryos.     

Detection of n-myc gene amplification in neuroblastoma using polymerase chain reaction based methods

We have used semiquantitative and real-time quantitative PCRs to detect n-myc gene-amplification in 21 frozen neuroblastoma biopsies and IMR 32 cell line in order to predict biological behaviour of the tumors. Two primer pairs were used in the semiquantitative method to co-amplify a 520-bp fragment of the beta -globin gene -used as a single copy reference standard -and a 258-bp fragment of the n-myc gene. After 30 cycles the PCR products were electrophoresed through an agarose gel and were compared to each other with use of a gel-densitometer. Real-time quantitative analysis was performed in a LightCycler instrument. A single primer pair was used to amplify a 120-bp fragment of the n-myc oncogene and a LC640-labelled fluorescent probe pair to detect the product. Calibration curve, which was set up from a serial dilution including samples with 1, 2, 10, 13, 25-fold n-myc oncogene amplification, was employed for quantitative analysis. Semiquantitative method did not show distinct difference between tumor groups with no amplification and less than 10-fold amplification, while quantitative LightCycler analysis was able to detect even 2-fold amplification. In situ PCRs were performed in two cases of differentiated tumor samples which contained n-myc amplification. We used biotinylated ATP labelling and the same primer pair as for the LightCycler analysis.In both cases differentiated cell forms did not show n-myc gene amplification, while considerable amplification was detected in the neuroblasts.     

Rapid detection and identification of odontoglossum ringspot virus by polymerase chain reaction amplification

Abstract A hemolysis gene ( hlx ) which lyses sheep erythrocytes on blood agar plates when expressed in Escherichia coli was cloned from Vibrio cholerae . The cloned gene is predicted to encode a polypeptide of 92 amino acid residues with a deduced molecular mass of 10451. E. coli transformed with this gene lysed sheep, goose, horse and chicken erythrocytes but not those of guinea pig and human. The hlx gene was observed in classical- and El Tor-biotype V. cholerae O1, V. cholerae non-O1, and V. mimicus , but not in V. parahaemolyticus .     

Recombination and amplification of multiple portions of genomic DNA by a modified polymerase chain reaction.

A novel method, a modified polymerase chain reaction (mPCR), is reported for in vitro recombination and amplification of two or more regions of DNA located in distinct parts of a genome such as exons of a gene. The reaction consists of three stages. In the first stage, the portions are amplified individually with the aid of four or more primers. Of the primers, two (outside primers) are complementary to 3'-terminal parts of the objective, recombinant DNA, and the others (inside primers) are complementary to 3'-terminal parts of the other portions. Some of the inside primers have extra nucleotide sequences at their 5'-termini which are complementary to 5'-terminal parts of the portions to be recombined. In the second stage, the strands with complementary sequences in their 3'-terminal parts are annealed sequentially to one another and are converted to recombinant DNA with DNA polymerase. In the final stage, the objective DNA is amplified with the excess outside primers. The application of this method for in vitro recombination and amplification of three exons of the gene for human muscle-specific phosphoglycerate mutase is described.     

DNA elution and amplification by polymerase chain reaction from dried blood spots.

A quick, sensitive and easily automatizable method for PCR amplification of genomic DNA eluted from dried blood spots is described. DNA is eluted from a 3-mm spot routinely used for neonatal screening of inherited diseases either by boiling or by sonication. A preliminary and brief spot-autoclaving step is mandatory to ensure optimal and reproducible PCR amplifications. Only 1% of the eluted DNA is required for PCR analysis allowing the execution of multiple genetic tests on the same blood spot. The method has been successfully applied to the detection of a known phenylketonuria-causing mutation and will facilitate the analysis of the genetic repository provided by Guthrie's cards stored in neonatal screening laboratories.