Second strand synthesis

Second-Strand Synthesis and Amplification by Error-Prone PGR... 

Use hot-start tubes and assemble the bottom and top part of the reaction for second-strand synthesis and amplification of the DNA template by error-prone PCR. Hot-start PCR is the PCR technique of assembling the reaction mixture at a temperature that is greater than the annealing temperature. This procedure increases precision, yield, and specificity. The pre-adhered wax bead assures synchronous reaction start-up and eliminates the need for using mineral oil. 

Second-strand synthesis Execute the following program on a PCR machine ... 

Second-strand synthesis and amplification by error-prone PGR mix... 

Ferrari, F. K., Samulski, T., Shenk, T. and Samulski, R. J. (1996). Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors. J. Virol. 70, 3227-3234. 

Extracellular barriers (DNA, enzymes, mucus) Paucity of receptors Proteosome-mediated degradation Inhibition of second-strand synthesis Airway clearance, anti-inflammatory, anti-protease pre-treatments Alternate serotypes, targeted capsid mutants Proteosome inhibitors (tripeptides, anthracyclines) Tyrosine kinase inhibitors... 

Because of the hairpin formation, these dyes are in such a close proximity that their fluorescence is quenched (molecular beacon Box 18) unless the structure is unfolded in the course of second-strand synthesis (Figure 4.3.4b). Thus, detection of a fluorescence signal from one of both dyes is a direct measure of the progress of the reaction. These researchers also showed that primer extension reactions can be monitored directly in cleared lysates of cells overexpressing the Klenow fragment of E. coli DNA polymerase I. Thus, the molecular beacon assay might supersede extensive purification. 

AAV transduction can occur in the absence of cell cycle however, transduction efficiency is markedly improved in cells in S-phase (22). Furthermore, activation of the cellular DNA repair machinery also supports second strand synthesis, thus improving AAV transduction (23,24). The latter suggests that transduction of terminally differentiated postmitotic cells may be hampered to some extent due to insufficient second strand synthesis. 

Two building blocks, derived from glycerol (27) and cis,cfs-l,3,5-cyclohexane-triol (28) have been prepared for the synthesis of ODNs with a 3 -3 linkage." The building blocks allow for either symmetric or asymmetric ODN synthesis for asymmetric synthesis, removal of the DMT group allows for first strand synthesis which is then capped with benzoyl chloride. Removal of the silyl protecting group then allows second strand synthesis. 

The result is high-quality arrays that can be used with a fluorescence detection system in a manner similar to the glass slide microarrays, although only single color detection is used. cDNA is synthesized from a test RNA sample by reverse transcription followed by second strand synthesis. This is then used as a template for an in vitro transcription reaction to produce biotin-labeled cRNA. As well as introducing the label for detection, this step permits amplification of the RNA and thus enables less starting material to be used. The cRNA is hybridized to the GeneChip and the biotin detected with an avidin-... 

Carry out second-strand synthesis by adding the following to 15 pL of tailed cDNA ... 

Construction of a subtracted cDNA library. Begin with Subheading 3.4.2., step 2—second-strand synthesis. 

Add reagents for the second-strand synthesis, mix by pipeting, and return the tube to the PCR block at 16°C (do not use heated lid at this point) for a further 2h. Add T4 polymerase to fill any gaps in the cDNA and continue to incubate at 16°C... 

When 2pg of total RNA is used as a starting amount, the whole first-strand reaction (20 J,L) is used as a template in the second-strand synthesis. If >10pg is nsed, then this volume is reduced to 10 pL. 

Cleavage of the hairpin loop with SI nuclease necessarily removes part of the DNA sequence corresponding to the S end of the mRNA. An improved method for full length duplex cDNA synthesis overcomes this problem. The first cDNA strand is tailed with oligo-dC so that second strand synthesis can be primed with oligo-dG (Hg.4). 

PE-RTbio 5 -biotin-TCCTGCTGAACCGCTCTTCCGATCT Second strand synthesis (Method 1)... 

The proper amount of RT-SE3n primer see Table 1) with oligo d(T) to anneal at the 3 end used in the second strand synthesis is to ensure enough end primer concentration for Klenow extension reaction [13]. 

FIGURE 6.1 Template specificity of E. colt DNA Pol 1 and Pol Ik. (a) Typical second-strand synthesis on a single-stranded DNA template containing a primer-annealed double-stranded region, (b) filling-in reaction on a gapped duplex, and (c) polymerization reactions on nicked DNA which, depending on polymerases, may be a nick translation Pol I, path A) or a strand displacement synthesis (Pol Ik, path B). 

When second-strand DNA is synthesized by the one-tube method (see below), first-strand cDNA synthesis is performed without actinomycin D and the first reaction mixture is directly adjusted with the reaction buffer for second-strand synthesis. When second-strand DNA is synthesized by the conventional procedures (see AMV RTase), the first reaction is terminated by adding 2 /zl of SDS (10%, w/v) and 2 ju.1 EDTA (0.5 M, pH 8.0), and the cDNA products are precipitated with ethanol. 

Sequenase is particularly useful for introducing mismatching nucleotides during the oligonucleotide-directed second-strand synthesis in the presence of a biased nucleotide pool (9). This forced misincorporation mutagenesis technique is useful for generating localized random mutants. 

In summary, a variety of barriers to eflScienttransduction of airways in vivo by luminal application ofvector exist Decreased binding and uptake of vector due to lack of apical membrane receptors and internalization pathways appears to be a common barrier to all vectors tested in clinical trials to date. Other barriers such as second-strand synthesis and nuclear entry are more vector specific. Thus, stiat ies that permit better apical membrane entry or that allow access of vector to the basolateral surface are likely to be generally useful. Circumventing the immune response will be necessary to alleviate safety concerns and to facilitate repetitive administration of transient expression vectors. 

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