Alternatively, some constructs were prepared by annealing of two overlapping oligonucleotides that were then filled in by PCR using Vent DNA polymerase (New England Biolab), producing a double-stranded DNA fragment.
Future work could augment the dataset by filling in population gaps using updated datasets, such as UN population datasets, archaeological data, or the work of Bairoch which has suggested systematically increasing the populations of European and Latin American cities in particular1,5,33.
The EcoRI and BamHI sites were eliminated sequentially by digestion, filling in the ends using Klenow enzyme, and religation which resulted in the intermediate vector pPZP650.3 (not shown).
The gaps in the assembly after high-throughput sequencing were filled in by direct sequencing using the ABI 3730 DNA sequencer by Sangon Biotech (Shanghai) Co., LTD., using adjacent PCR primers.
After the DNA purification the 3'-5' adaptor strands were filled in by nick-translation using a dNTP solution, containing 5m-dCTP instead of dCTP as previously described [15].
Parental education level was assessed individually in the questionnaire filled in by the parents, using the question: " What level of education do you have?".
The tools were specially designed forms comprising check lists and rating scales to be filled in by the investigators using direct observation.
Standardized questionnaires filled in by parents were used to collect information on health outcomes and exposure to traffic among 33,632 6–7 and 13 14 years old children and adolescents.
The DDHQ was filled in by adolescents and was used to assess frequency of daily hassles concerning ill parents (6 items, α = 0.61) and frequency of daily hassles concerning healthy parents (5 items, α = 0.71).
