By Natalie Betz, Ph.D.
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| Blood Volume Used (µl) | ||||
| Reagent | 20 | 50 | 100 | 300 |
| Cell Lysis Solution | 60µl | 150µl | 300µl | 900µl |
| Nuclei Lysis Solution | 20µl | 50µl | 100µl | 300µl |
| Protein Precipitation Solution | 6.7µl | 16.5µl | 33µl | 100µl |
| Isopropanol | 20µl | 50µl | 100µl | 300µl |
| DNA Rehydration Solution | 10µl | 25µl | 50µl | 150µl |
All volumes of blood were processed in duplicate (50µl, 100µl and 300µl) or triplicate (20µl). Aliquots of blood were added to tubes containing the appropriate amount of Cell Lysis Solution, inverted to mix and incubated for 10 minutes at room temperature to lyse the red blood cells. The samples were centrifuged at 13,000-16,000 x g for 20 seconds, and as much of the supernatant as possible was discarded. The tubes were vortexed for 10-15 seconds, and the Nuclei Lysis Solution was added. Following lysis of the white blood cells, the Protein Precipitation Solution was added, and the mixture was vortexed. The samples were centrifuged at 13,000-16,000 x g for 3 minutes. The supernatant was transferred to a clean tube containing room temperature isopropanol. The DNA was pelleted by centrifugation at 13,000-16,000 x g for 1 minute, washed once with room temperature 70% ethanol and air-dried for 10-15 minutes. DNA Rehydration Solution was added, and the samples were incubated at room temperature for 20 minutes or at 4°C overnight.
PCR Amplification(a): Samples to be amplified were purified in duplicate. One sample of each blood volume was rehydrated at room temperature for 20 minutes, while the remaining sample was rehydrated at 4°C, overnight. An aliquot of each sample was analyzed by spectrophotometry at 260nm (A260). A 1.8kb fragment from the adenomatous polyposis coli (APC) gene, segment 2, was amplified from each sample using either 20ng of DNA (based on the A260 reading) or 5µl of the sample. PCR was performed in a final volume of 50µl and contained: 2.5 units Taq DNA Polymerase, 1X thermophilic buffer, 1.5mM MgCl2, 200µM each dNTP, 50pmol each for APC segment 2 specific oligonucleotide primers. Amplification was performed in a Perkin-Elmer 480 thermal cycler using the following profile: 1 cycle of 95°C for 5 minutes, 35 cycles of 94°C for 30 seconds, 55°C for 1 minute, and 72°C for 2 minutes, followed by 1 cycle of 68°C for 8 minutes and soaking at 4°C.
Gel Electrophoresis: For those samples analyzed only by agarose gel analysis, duplicate samples were processed. To one sample of each blood volume, 2µl of 5mg/ml linear acrylamide (Ambion) was added as a carrier following nuclei lysis. For 20µl sample volumes, two sets of triplicate samples were processed and the DNA from each set of triplicates was pooled following rehydration. Each sample (10µl) was analyzed by 0.9% agarose gel electrophoresis (in 1X TAE). The DNA was visualized by ethidium bromide staining following electrophoresis.
For analysis of the PCR-generated fragments, a 1% agarose gel and 6µl of each sample was used. The DNA was visualized as described above.
The Wizard® Genomic DNA Purification Kit has the ability to isolate genomic DNA from small volumes of blood in microcentrifuge tubes. To demonstrate this, DNA was isolated from decreasing volumes of fresh whole human blood using microcentrifuge tubes and 20 to 300µl of blood.
Acrylamide Carrier: Genomic DNA was isolated from 20, 50, 100 and 300µl of whole blood in duplicate, with one of the samples receiving linear acrylamide as carrier following the nuclei lysis step. The resulting purified DNA was rehydrated overnight at 4°C, and 10µl of each sample were analyzed on an agarose gel. As seen in Figure 1, high molecular weight genomic DNA was visible from each of the whole blood samples, even when as little as 20µl of blood was used in the purification. The presence of linear acrylamide as carrier did not appear to significantly affect DNA yield, and thus is not necessary, even with small sample volumes.
Figure 1. Genomic DNA isolated from decreasing volumes of whole blood, in the presence (+) or absence (-) of linear acrylamide carrier. Lane M contains 2µl Lambda/EcoR I Markers (Cat.# G1721); lane 1, 100ng Human Genomic DNA (Cat.# G3041) positive control (C); Lanes 2 and 3, 300µl; lanes 4 and 5, 100µl blood; Lanes 6 and 7, 50µl blood; lanes 8 and 9, 20µl blood; and lane 10, 300ng Human Genomic DNA (Cat.# G3041) positive control (C). Lanes 2, 4, 6 and 8 were isolated in the absence of carrier (-). Lanes 3, 5, 7 and 9 were isolated in the presence of carrier (+).
For PCR amplification, duplicate samples were processed. One sample was rehydrated for 20 minutes at room temperature while the other sample was rehydrated overnight at 4°C. (Table 1 shows Rehydration Solutions Volumes.) Prior to PCR amplification and following rehydration, the absorbance at 260nm was measured for each sample (Table 2).
Table 2. Yield of Genomic DNA Isolated From 20 to 300µl of Whole Blood Using Two Rehydration Methods.
| Yield (µg/ml) | ||||
| 300µl | 100µl | 50µl | 20µl | |
| 20 minutes, room temperature |
0.405 | 0.111 | 0.036 | 0.033 |
| overnight, 4°C |
0.066 | 0.03 | 0.015 | 0.045 |
Rehydration for 20 minutes at room temperature appeared equivalent to, if not better than, rehydration overnight at 4°C. However, the differences could simply be due to variance in yield between the duplicate samples, and not differences in rehydration efficiencies. Following rehydration, a 1.8kb fragment from segment 2 of the APC gene was amplified from the purified DNA, using either 20ng of DNA (based on the A260 reading) or 5µl of each DNA sample. The results, shown in Figure 2, illustrate that the expected product can be amplified from either 20ng or 5µl of each sample; however, the amplification yield from 20ng of DNA isolated from 20µl of blood is significantly less than from the other volumes. This decrease in amplification is probably because accurate DNA concentration estimations using A260 absorbance values are not possible at such dilute DNA concentrations. Therefore, when amplifying DNA isolated from 20µl of blood, we recommend using 5µl of the DNA in PCR. This eliminates the need to waste any of the sample by taking an absorbance reading.
Figure 2. PCR analysis of genomic DNA isolated from decreasing amounts of whole blood. Panel A: Amplification products from PCR using 20ng of genomic DNA as a starting template. Panel B: Amplification products from PCR using 5µl of the isolated genomic DNA as the starting template. Lanes M, 1kb DNA Ladder (Cat.# G5711); lanes 1, no DNA control (–); lanes 2, Human Genomic DNA (Cat.# G3041) positive control (+); lanes 4 and 5, genomic DNA from 300µl blood; lanes 6 and 7, 100µl blood; lanes 8 and 9, 50µl blood; and lanes 10 and 11, 20µl blood. Lanes 3, 5, 7 and 9 contain samples that were rehydrated at room temperature for 20 minutes. Lanes 4, 6, 8 and 10 contain samples rehydrated at 4°C overnight.
These results indicate that the Wizard® Genomic DNA Purification Kit can easily be scaled for volumes of blood from 300 to 20µl. The reactions for even the smallest volumes can be performed in microcentrifuge tubes and still produce acceptable quantities of high-quality genomic DNA. No special precipitation or rehydration conditions are required to gain acceptable yields of genomic DNA, and the DNA is suitable for many applications including PCR amplification.
(a)The PCR process is covered by patents issued and applicable in certain countries. Promega does not encourage or support the unauthorized or unlicensed use of the PCR process. Use of this product is recommended for persons that either have a license to perform PCR or are not required to obtain a license.
Wizard is a trademark of Promega Corporation and registered with the U.S. Patent and Trademark Office.
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