The study was ethically approved by Institutional Ethics Review Board (IERB) of St. John’s Medical College and Hospital, Bangalore (India) with approval number IERB/1/77/05. After explaining the study to participants, informed written consent (as per norms of Indian Council of Medical Research (ICMR) Government of India) was obtained from volunteer participants.

This was part of a multicenter study involving the Centre for Global Health Research (CGHR) Bangalore unit and Tata Memorial Centre, Mumbai unit, who worked together to conduct study DBS. DBS samples were collected at health checkup camps in rural and urban areas of Bangalore city through a sample registration system (SRS). 3000 DBS samples were prepared through finger prick method by using lancet (Accu Chek Softclix Lancet, Roche), puncture the finger site using lancet, drop of blood form which is lightly touch the circle of filter paper cards (GE Health Care Life Science, Catalog no. 10534612) and form valid DBS during health checkup camp by CGHR at Bangalore unit and transported to Tata memorial Centre (TMC) Mumbai for further analysis. Samples were collected between the years 2005–2007. DBS samples were collected by trained staff. Systematic random samples (n=40) were selected from top to bottom order from collected DBS. The following anthropometric measurements were recorded; height, weight, waist-to-hip ratio, blood pressure with gender and age. The complete study was explained to the subjects, and only voluntary participants aged between 18–49 years were included in this study after obtaining written informed consent as per the norms of Indian Council of Medical Research (ICMR) Government of India.

Finger prick blood DBS Preparation. Finger prick blood was collected using a lancet to puncture the fingertip. Once a full small drop of blood is formed it was lightly touched to the center of the circle on the filter paper (GE Health Care Life Science, Catalog no. 10534612) to form valid DBS. These were collected from study participants during health checkup organized at government schools, and at the center of villages. Cards were dried for 2 hours in velcro rack and packed in sealed ziplock bag with 1–2gm desiccant sachet ( Figure 1). Only valid DBS samples were used for gDNA extraction determined by the blood sample completely saturating each circle on the card, and not overlapping or merging with other blood circles. Prepared DBS samples were transported to the laboratory at the Centre for Cancer Epidemiology, Tata Memorial Centre, where they were stored in a -80°C refrigerator.

We have used only good quality DBS samples for gDNA extraction, it is defined as the complete saturation of whole blood over the complete circle of blood collection card ⁹ , blood card should be labelled and blood should adsorb on both side of the card. We have used only valid samples for gDNA extraction ( Figure 2).

A generic single hole 6mm punch plier was used to cut the blood spots from the Whatman 903 paper card. A punch of diameter 6mm represents approximately 8.7±1.9µl of blood spotted ¹⁰ . 1 – 4 blood spots of size 6 mm punch was added to an eppendorf tube and incubated with 200µl PBS (readymade PBS buffer used with pH 7.4 supplied by Gibco with Ref No. 10010-023) overnight at room temperature. Our major aim was to extract the maximum amount of gDNA, therefore we have used 6mm × 1 spot to 6mm × 4 spots for extracting gDNA from DBS ( Table 1). We have used phosphate buffer saline (PBS) for extraction of completely dry blood matrix on Whatman 903 for easy gDNA extraction, because once the surface of blood spot becomes wet, it is easy to extract DNA from the adsorbed blood.

We have applied 2 methods for gDNA extraction from DBS. ( 1) Column based (QIAamp DNA kit, Qiagen) ( 2) Magnetic bead based (ChargeSwitch Forensic DNA Purification Kit, Invitrogen).

Column based gDNA extraction from DBS. We used the QIAamp DNA kit (Qiagen, Catalog no. 56304). 1 – 4 blood spots 6mm in size were added with 180 µL of cell lysis buffer ATL (Lysis buffer supplied with Qiagen kit), and incubated in a waterbath (Trishul Equipment, Sr. No. 5460311) at 85°C for 10 min. 20 µL Proteinase K was added and incubated it at 56°C for 1 hour to denature the proteins. 3–4 µL RNAse was added immediately after to degrade RNA, then 200 µL buffer AL (Lysis buffer supplied with Qiagen kit) was added & mixed thoroughly by vortexing and incubated at 70°C in a waterbath (Trishul Equipment with Sr. No. 5460311) for 10 min. Buffer AL helps in complete cell lysis and binding of gDNA with the silica gel of the column provided in the Qiagen kit. gDNA was then immediately precipitated by adding 200 µL of 70% v/v ethanol. The solution was then transferred into a spin column (supplied with Qiagen kit) and centrifuged (Eppendorf 5810R) at 8000 rpm for 1 min. The spin column has the capacity to load approximately 600 microliter sample at a time, but generally we had approximately 1.2 or 1.4 ml of solution, we therefore performed the process 2–3 times. In this process gDNA becomes bound with the column, impurities are then washed out with 700 µL buffer AW1 (Wash buffer with a low concentration of quinidine) followed by 700 µL of AW2 (Wash buffer with Tris based ethanol solution used for removal of salts) buffer. Buffers AW1 & AW2 remove unwanted impurities from the gDNA. The empty tube was then centrifuged at 14000 rpm for 3 min for complete removal of ethanol from the gDNA. Finally the gDNA was eluted with 30 µl of pre-incubated elution buffer (AE).

Magnetic bead based gDNA extraction from DBS. We used the ChargeSwitch Forensic DNA Purification Kit (Thermofisher, Catalog No. CS11200). Processed DBS samples were added to 1ml lysis buffer with 10 µL of Proteinase K in a tube, vortexed, and incubated at 55°C in a waterbath (Trishul Equipment Sr. No. 5460311) for 1 hour. Blood spots were removed after complete cell lysis and 200 µL of purification buffer added, followed by 20 µL magnetic beads. The solution was then gently mixed, left for 5 minutes, and then incubated on a Magna Rack (Thermofisher, catalog no. AM10027) for 1 minute. The supernatant was removed after complete binding of the pellet to the magnet of the Magna Rack. The pellet containing gDNA was then washed with 500 µL wash buffer (W12) 3 times and finally DNA eluted with 30–60 µL Elution Buffer (E5). We have followed the protocol as per manufacturer recommendation with some modifications; incubation time was increased from 4 hours to overnight for complete extraction of eluate from filter paper, and we also increased the time for proper binding of gDNA with the spin column.

Concentration of extracted gDNA was measured using a Qubit 3.0 fluorometer and purity was measured by a spectrophotometer. Quality and integrity of gDNA was checked by performing a 0.8% Agarose gel electrophoresis.

Qubit 3.0 Fluorometer (Thermofisher, Catalog No. Q32850) was used to measure gDNA concentration by taking 1 µl gDNA sample in 0.2ml PCR tube with 200 µl buffer (199µl dsDNA BR buffer + 1 µl Ethidium Bromide dye) after proper mixing for 1min. Concentration was then measured with a fluorometer.

A spectrophotometer at wavelength 260/280 ratio (NanoDrop 2000 Thermofisher) was used to check purity of gDNA, by applying 1 µl gDNA sample directly to the device and measuring the 260/280 ratio.

Agarose gel electrophoresis was performed following preparation of a 0.8% agarose gel which was loaded with 5 µl gDNA in wells and run at 70–80 volts for 2 hours. A gel photograph was captured using a gel dock (Model: UVP EC3-Imaging System).

We obtained average gDNA concentrations of 6mm × 1 spot to 6mm × 4 spots from DBS cards ( Table 1). In our findings, we observed large variation in the concentration, from 2.16ng/µl to 24ng/µl, of the extracted gDNA ( Table 2). The integrity of gDNA was checked using 0.8% agarose gel electrophoresis, and highly intense single bands were observed on the gel ( Figure 3). The purity of gDNA was measured at an absorbance of 260/280nm (1.8–2.0) ratio. If it is less than 1.8 or greater than 2.0 it indicates the presence of impurities in the genetic material. gDNA concentration of each DBS with different number of blood spots, with their purity and total gDNA yield are presented in Table 3.

This amount of gDNA extracted can be used for polymerase chain reaction (PCR), and PCR based molecular assays such as PCR based sequencing, PCR based genotyping, but can’t be used for whole genome sequencing or genotyping ¹¹ .