DNA barcoding surveys of small insects usually extract DNA from either a complete insect or a leg. Little is known about how to optimize DNA quantity and quality from different insect parts while preserving a morphological voucher. Here, we quantify DNA yield from different body parts (antenna, hind leg, forewing, hind wing and abdomen) of the micro-moth Cameraria ohridella (Lepidoptera: Gracil...

A new method to produce hot-start conditions in PCR is described. Short double-stranded DNA fragments were found to inhibit the activity of DNA polymerases from Thermus aquaticus and Thermus flavus. This inhibition is not sequence specific, but exclusively dependent on the melting temperature of the fragments as shown by its correlation to their melting curves as measured. This property is expl...

PCR Conditions: The PCR reaction is carried out in a total volume of 50 /*1 containing approximately 250 ng DNA, 2 units Taq DNA polymerase, 50 pmol of each primer, 200 /M dNTP's, 10 mM Tris-HCl pH 8.3, 50 mM KC1, 1.5 mM MgCl2, 0.001% gelatin. The amplification is performed for 30 cycles with an annealing temperature of 60°C. The amplified product is digested with TaqI and the DNA fragments are...

Methods are presented for the improved yield and analysis of blunt-ended cloning of PCR-generated DNA fragments. We show that Pfu DNA polymerase polishing of Taq DNA polymerase-generated fragments increases the yield and efficiency of cloning. Using a triple primer set consisting of two outside, asymmetrically distanced primers and one fragment-specific primer, both the presence and orientation...

We describe compartmentalized self-replication (CSR), a strategy for the directed evolution of enzymes, especially polymerases. CSR is based on a simple feedback loop consisting of a polymerase that replicates only its own encoding gene. Compartmentalization serves to isolate individual self-replication reactions from each other. In such a system, adaptive gains directly (and proportionally) tr...

The Thermus aquaticus DNA polymerase I (Taq Pol I) gene was cloned into a plasmid expression vector that utilizes the strong bacteriophage PL promoter. A truncated form of Taq Pol I was also constructed. The two constructs made it possible to compare the full-length 832-amino-acid Taq Pol I and a deletion derivative encoding a 544-amino-acid translation product, the Stoffel fragment. Upon heat ...

Genomic DNA isolated from archived paraffin-embedded tissues (PETs) has important applicability in genetic epidemiological studies. To determine the accuracy of the sequence data, using DNA derived from PET among patients with known mutations characterized from blood, we conducted a blinded factorial experiment to simultaneously examine the influence of mutation type, age of the PET, PCR produc...

range of blood-cell concentrations (between 0.125–1.0 μL of blood; see References 5 and 6). One difference between the original vs. the modified method is degradation of hemoglobin, or heme compound, by boiling in NaOH that results in a stronger inhibitory effect on the Taq DNA polymerase (2,4). In addition, it is possible that repeated boiling of the sample before DNA amplification increases t...

The Thermus aquaticus DNA polymerase I (Taq Pol I) gene was cloned into a plasmid expression vector that utilizes the strong bacteriophage lambda PL promoter. A truncated form of Taq Pol I was also constructed. The two constructs made it possible to compare the full-length 832-amino-acid Taq Pol I and a deletion derivative encoding a 544-amino-acid translation product, the Stoffel fragment. Upo...