DNA Amplification and Sequencing

PCR contained the following components 1 to 6 il of DNA extract, 5.0 pi of lOx MgCl2 + PCR Buffer n (Perkin-Elmer AppUed Biosystems), 1.0 pi of 10 mM dNTPs, 0.5 pi of each primer pair at 25 pM, 0.25 pi of AmpUtaq Gold DNA Polymerase (Perkin-Elmer) and dH20 to a total volume of 50 pi. For most extractions, 1 to 4 pi of DNA yielded visible PCR product. The most frequently successful strategy when PCR reactions failed was to use 2 pi of 10- to 50-fold diluted extract. 

AU PCR reactions were done with a GeneAmp 9600 thermocycler (Perkin-Elmer) or DNA Engine thermocycler (MJ Research). Each PCR reaction was preceded by 10 to 12 min at 95C (a 

Voucher Specimens and GenBank Accessions for Sequences Used in this Study 

OTU/Taxon (Herbarium) Locality Information atpB rbcL rps4 trnL 

Anacamptodon splachnoides (Froel. ex Brid.) Brid. M. L. Sargent s.n. (UC-Culture) U.S., Indiana — AF231077 — — 

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Biological identification DNA amplification and sequencing, optical sensing, lab-on-chip and portable systems... 

DNA-based approach (phylogenetic approach) has been reported by Canadian investigators for various species of psilocybin mushrooms which are quite helpful in the identification of species that contain the psychoactive compounds [153]. The method includes DNA amplification and sequencing of the internal transcribed spacer region of the rDNA (ITS-1) and a 5 portion of the nuclear large ribosomal... 

A site at the Agricultural Experimental Station (Ithaca, NY) was treated in microcosms with C-labeled glucose, phenol, caffeine, and naphthalene. Levels of C02 were measured to assess utilization of the substrates, and the populations analyzed by separating the C-labeled DNA by density centrifugation, followed by PCR amplification and sequencing of 16S rRNA (Padmanabhan et al. 2003). Populations contained relatives to a range of bacteria that varied with the substrate. Only relatives of Acinetobacter were found in all samples, and for caffeine only Pantoea. 

The procedure for epitope mapping by the phage display method involves biopanning, amplification, assay for positive colonies, DNA isolation, and sequencing. Some steps of the procedure must be performed at the same time. Therefore, we describe the protocols in Subheadings 3.1. and 3.2. on a day-by-day manner. All steps of biopanning and phage amplification should be carried out under sterile conditions, when possible. 

Wilson, M. R., Polanskey, D., Butler, J., DiZinno, J. A., Replogle, J., and Budowle, B. (1995b). Extraction, PCR amplification and sequencing of mitochondrial DNA from human hair shafts. Biotechniques 18, 662-669. 

Bisulfite conversion followed by PCR amplification and sequencing has become the gold standard in determining patterns of DNA methylation. This method excels over all the others because it allows information about methylation at the base-pair resolution. Nevertheless, until recently, its utilization genome-wide has been slow because bisulfite-based methods are not easily adapted to the array-based hybridization approaches. Yet, bisulfite-converted DNA has been rapidly adapted to cheaper everyday deep-sequencing techniques (Figure 3B). 

El. Eakin, A. E., Bouvier, J., Sakanari, J. A. Craik, C. S., and McKerrow, J. H., Amplification and sequencing of genomic DNA fragments encoding cysteine proteases from protozoan parasites. Mol. Biochem. Parasitol. 43, 1-8 (1990). 

Multiple PCR chambers have been fabricated on a single microfluidic chip and explored for high throughput PCRs [78-83]. An example of a multichamber micro-PCR device, the micro-DNA amplification and analysis device, (p-DAAD) consisted of 16p-DAADs in parallel with each p-DAAD consisting of four microreactors fabricated on a 4" silicon wafer (see Fig. 4). Multichamber micro-PCR devices [84] have been demonstrated for DNA amplifications of five gene sequences related to E. coli from three different DNA templates and detected by TaqMan chemistry with a limit of detection (LOD) of 0.4 copies of target DNA. 

Figure 3.18 PCR mutagenesis. This is the simple way to engineer the primary structure of a protein of interest. Where desired mutations are near the 5 -terminus or the 3 -terminus or the sense strand, then the mismatched primer technique is used. In the illustrated case, the desired mutation is near the 3 -terminus so a normal sense strand primer is combined with a mismatched complementary strand primer containing a mutation (blue). When the PCR reaction is allowed to proceed with the template DNA, then the mismatch in the complementary strand primer forces a mismatch to appear in both sense and complementary strands of the final PCR amplification product resulting in a PCR mutant gene. Restriction cutting and ligation of the mutant PCR product into a cloning or expression vector generates a mutant recombinant pDNA construct ready for transformation and selection, then DNA purification and sequencing of correct mutant recombinant DNA.

In the mid 1970 s techniques for the rapid sequencing of DNA were developed but it was not until 1985 when DNA amplification by the pol3rmerase chain reaction (PGR) was devised (Saiki et al. 1985, Mullis et al. 1986) that sequence analysis of nucleotides became feasible on a large scale. PGR allows the rapid selection, isolation, amplification and sequencing of DNA regions of interest from small amounts of tissue (Simon et al., this volume) providing data on mutations at the nucleotide level which are extremely valuable for systematic studies. 

Kocher TD, Thomas WK, Meyer A, Edwards SV, Paabo S, Villablanca FX, Wilson AC (1989) Dynamics of mitochondrial DNA evolution in animals Amplification and sequencing with conserved primers. Proc Natnl Acad Sci USA 86 6196-6200... 

Kocher, T.D., Thomas, W.K., Meyer, A., Edwards, S.V., Paado, S., Villablanca, F.X., and Wilson, A.C., Dynamics of mitochondrial DNA evolution in animals amplification and sequencing with conserved primers, Proc. Natl. Acad. Sci. USA, 86, 6196-6200, 1989. 

Non-Newtonian liquids are used in numerous microfluidics applications, including microscale viscosity and rheology measurements, amplification and sequencing of DNA, fundamental investigations of elastic flows, and development of fluidic memory and control devices. Although these applications span a wide range of flow conditions and non-Newtonian fluid properties, similar experimental methods are used. In this section we summarize some of the experimental... 

PCR is being used in phylogenetic and evolutionary studies. PCR amplification and screening of ribosomal RNA (rRNA) genes and mitochondrial DNA have been used to estimate relationships between species and subspecies. Besides studies on the relationships of contemporary species, PCR allows studies on extinct species because it can be performed on material in museum collections. Amplification and sequencing of mitochondrial DNA are also used in evolutionary studies. 

Chiang, T. L, Schaal, B. A. and Peng, C.-l. (1998) Universal primers for amplification and sequencing a noncoding spacer between the atpB and rbch genes of chloroplast DNA. Botanical Bulletin of Academia Sinica, 39 245-250. 

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