Fill-in reactions

Fill-in reactions, relatively low specific activity, may be useful for specific labeling of oligomers... 

Fig. 7.16. T4 DNA polymerase (I) without dNTPs behaves as an exonuclease. After a convenient time span, dNTPs (with label) can be added to obtain partly labeled probes. Restriction and purification yields strand-specific probes. A similar approach can be taken by exonuclease and fill-in reactions.

Reverse transcriptase is especially effective for small fill-in reactions. 

The 3 -ends are labeled, after specific restriction enzymes have produced a protruding 5 -end, by a fill-in reaction that allows only one label to be introduced (e.g., labeling with dATP with an overhang with 2 Ts should be avoided). Reverse transcriptase or Sequenase are the preferred enzymes for this fill-in although the Klenow fragment has been widely used. The 3 -> 5 exonuclease activity of Klenow, however, may remove the protruding template end. 

Repair synthesis is executed by components of the replication machinery-the clamp loader RFC, the processivity factor PCNA, and the replicative Pols 8/e [51, 94], Recent studies have additionally implicated the translesion synthesis Pol k in repair synthesis [95], but what role the different polymerases play in the process remains to be elucidated. Following the fill-in reaction, the nick in the DNA is sealed by XRCC1-DNA ligase 3a in all stages of the cell cyde and by DNA ligase 1 in proliferating cells [96], thus completing the NER process. 

FIGURE 2 Synthesis of the labeled DNA primer by the filling-in reaction with Sequenase version 2. 

In the final assembly reaction, the VH-link and link-VK DNA are joined into a single chain Fv antibody fragment and restriction sites are added. These restriction sites are used to clone the scFv into a phagemid vector as a Sfil-Notl DNA fragment. The primers contain either Sfil (Mouse VH Back Sfil primers) or Notl (Mouse JK Forward Notl primers) restriction sites. They anneal to the 5 end of the heavy chain (Sfil restriction site) or the 3 end of the li t chain (Notl restriction site). Because of the complementarity of the linker DNA between the two PCR fragments, a fill-in reaction is primed in the presence of AmpliTaq DNA polymerase (see Figure 1C). For this reaction to proceed efficiently, approximately... 

A ds oligonucleotide (with 5 overhang or with blunt end) can be endlabeled efficiently using T4 polynucleotide kinase. Oligonucleotides with 5 overhang can also be labeled by fill-in reaction with Klenow enzyme. 

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). 

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