Figure 4

Application of human genomic DNA with pUC19 as Stuffer DNA. (a) The schematics of the idea of Stuffer DNA. 1-In case of much diluted samples, relegation preferentially leads to self-circularization of fragments, which are then amplified as individual molecules. 2-The addition of Stuffer DNA derived from an unrelated source to the sample DNA favors intermolecular ligation and leads to the formation of long concatamers, both linear and circular. These long concatamers can then be reliably amplified using MDA technique. (b) Amplification of hgDNA with stuffer DNA using MDA technique. Two different amounts of hgDNA were taken (1:10 ng/μ L and 2:1 ng/μ L) and a fixed amount of pUC19 (100 ng) was used as stuffer DNA. The amplification products are indicated by asterisks. 0.8% agarose gel stained with SYBR Green was used for electrophoresis. (c) Re-digestions of samples [previously digested with AseI and MseI (both gives identical cohesive ends), religated and amplified], with the same enzyme (MseI) and a different enzyme (HpaII). Odd numbers represent the samples redigested with MseI, while even numbers represent HpaII digests (1,2: 10 ng and 3,4: 1 ng). Asterisks indicate HpaII fragments that do not contain internal MseI sites and thus are preserved after MseI digestion and religation. 10% polyacryamide gel stained with EB was used for electrophoresis. (d) q-PCR data of 10 ng and 1 ng hgDNA samples MDA amplified with stuffer DNA. 6 different primer pairs were used for quantitative analysis. As a template for q-PCR, native hgDNA (left graph), and the MDA-amplified DNA (10 ng – middle graph, 1 ng – right graph) were used. Shown are C _T values calculated for each fragment according to Materials and Methods. (e) To see if the amplification preserves the proportions among different genomic regions, for each MDA experiment, ratios were calculated between C _T values of different fragments, taking P1-hgDNA as reference for hgDNA control samples and P1-MDA for MDA processed samples. Data for P6-MDA in case of 1ng input is not shown, as it did not give the correct amplification product.