IV. Results
4.1 Biotin-labeled DNA incubated with streptavidin coated magnetic beads
The pGEM-URA3 plasmid was digested into 3.3 kb and 1 kb short DNA fragments with 5’ end overhang by BspHI as shown in the band A and B of Figure 4B, and the band C in lane 2 was undigested pGEM-URA3 plasmid. The 3.3 kb and 1 kb fragments were named BspH-URA3-A and BspHI-URA3-B and were used as the short template DNAs.
The 4 bp 5’ overhang of the BspHI-digested URA3 (BspH-URA3-A and BspHI-URA3-B) were filled in with biotin-labeled dATP and non-biotin-labeled dNTPs by Klenow. The control was filled in with non-biotin-labeled dNTPs only, after that the fill-in products were cleaned up by ethanol precipitation twice.
4.1.2 Obtaining long DNA templates
For the long DNA template, the 12 bp 5’ overhang of λ DNA was filled in with either biotin-labeled dATP or dCTP mixed in non-biotin-labeled dNTPs (with the ratio 1:2) by Klenow. Some were filled in with non biotin-labeled dNTPs as control, and then the products were cleaned up by ethanol precipitation twice.
The fill-in λ DNA was then subjected to BaeI digestion to examine whether the fill-in of λ DNA was successful. Because of the 12 bp complementary 5’ end overhang, some of the λ DNA will form circular form DNA spontaneously. Only if the sticky end is filled in and
turned into blunt end, will the λ DNA become linear form. There are 10 BaeI cutting sites on λ DNA. For a linear form λ DNA after BaeI digestion, there will be 11 digestion fragments. The sizes of the fragments are 694, 6971, 5602, 2508, 496, 2461, 377, 2670, 22197, 4409, 117 bps.
If the λ DNA is in circular form, then after digestion, some of the 694 bps and 117 bps fragments will appear as 801 bps fragment (shown in Figure 5A). Therefore the appearance of the 801 bps DNA fragment can be the maker to monitor the Klenow fill-in reaction.
The 5’ overhang of λ DNA was filled in with biotin-labeled dATP or dCTP and mixed in non-biotin-labeled dNTPs and incubated with Klenow for 15, 30, 60 minutes. The mixtures without ligation reaction were then digested by BaeI in 25 for 2 hours. The digestion result ℃ was shown in Figure 5B. Lane 10 was the control λ DNA without nucleotides fill-in, and it had the 801 bps DNA fragment (shown in the band D of Figure 5B, indicated by an arrow) consisting of the 117 and 694 bps DNA fragments. Lanes 1-3 were the λ DNAs filled in with biotin-lableled dATP mixed with non-biotin-labeled dNTPs (BioA-λ DNA) and incubated in 25 for 15, 30, 60 minutes℃ . Lanes 4-6 were λ DNAs filled in with biotin-labeled dCTP mixed with non-biotin-labeled dNTPs (BioC-λ DNA) and incubated for 15, 30, 60 minutes.
Lanes 7-9 were the λ DNAs filled in with non-biotin-labeled dNTPs (dNTP-λ DNA) for 15, 30, 60 minutes. Gel electrophoresis result of lanes 1-9 reveals that they only had the 117 and 694 bps DNA fragments and did not have the 801 bps DNA fragment. Therefore, majority of the DNAs were filled in successfully.
4.1.3 Dot blot of biotin-labeled DNA
To further examine whether the biotin-labeled nucleotides were filled in, the reaction products were tested to determine whether they could be captured by Streptavidin-Peroxidase Polymer and the presence of peroxidase polymer was analyzed by dot blot. If the biotin-labeled nucleotides were filled in, then the Streptavidin-Peroxidase Polymer can attach to the biotin-labeled DNA. Therefore, the peroxidase polymer will be detected by dot blot.
There were four treatments in the dot blot of biotin-labeled BspHI-digested URA3: The DNAs of BspHI-digested URA3 were treated with either biotin-labeled dATP mixed with non-biotin-labeled dNTPs (BioA-BspHI-URA3) or non-biotin-labeled dNTPs (dNTP-BspHI-URA3) only. Biotin-labeled dATP with no DNA (BioA) and HindIII-digested λ DNA with biotin-labeled (Control DNA) were used as controls. As the result shown in Figure 6A, BspHI-URA3-BioA and control DNA appear to be positive on film, and the sizes of the dots reduced as the quantity of DNA decreased.
Next, the long DNA tempate, λ DNA was tested with the same treatments. The data in Figure 6B indicate that the λ DNA with either biotin-dATP (BioA-λ DNA) or dCTP (BioC-λ DNA) fill-in for various incubation times are positive and the λ DNA with non-biotin-labeled dNTPs fill-in (dNTP-λDNA), biotin-dATP with no DNA (BioA) and biotin-dCTP with no DNA (BioC) are not.
4.1.4 Analysis with the gel electrophoresis of the supernatant of the biotin-labeled DNA interacting with streptavidin coated magnetic beads
To determine whether the biotin-labeled DNA can be anchored to magnetic beads via the streptavidin coating, it is necessary to check the quantity of DNA within the supernatant after it was incubated with streptavidin coated magnetic beads (SA). If the DNAs can now be anchored to the SA, then they will be precipitated down and disappeared from the supernatant.
The biotin-labeled DNA was incubated with either 100 µg SA or 100 µl ddH2O at room temperature for 1 hour, and then the supernatant was analyzed by gel electrophoresis.
The results of BioA-BspHI-URA3 and dNTP-BspHI-URA3 incubated either with SA or ddH2O can be seen in Figure 7A. They reveal that the 1 and 3.3 kb DNA fragments of BioA-BspHI-URA3 in the supernatant decreased after incubated with SA compared to ddH2O(Figure 7A, bands E and F in lanes 1 vs. 2). And the supernatant of dNTP-BspHI-URA3 incubated with SA was almost the same as incubated with ddH2O
(Figure 7A, bands E and F in lanes 3 vs. 4). The 1.5 kb λ DNA PCR product is the internal control (band G).
As the Figures 7B shows, the band of BioA-λ DNA incubated with SA (BioA-λ DNA-SA) and that of BioC-λ DNA incubated with SA (BioC-λ DNA–SA) were relatively diminished compared with that incubated with ddH2O (Figure 7B, Band H in Lane 1 vs. 2 and Band H in Lane 3 vs. 4). The decreased quantity of BioC-λ DNA was more than that of BioA-λ DNA. The bands of dNTP-λ DNA incubated with either SA or ddH2O were almost the same (Band H in Lane 5 vs. 6). The 1.5 kb λ DNA PCR product is the internal control (Band I).
4.1.5 Dot blot of biotin-labeled DNA with SA after several washed
The biotin-labeled BspHI-URA3 and λ DNA were incubated with SA at room temperature for 1 hour and washed by 0.5 M NaCl in 1X TE buffer 4 times. Finally the reaction was resuspended in 200 µl 0.5 M NaCl in 1X TE. If one end of the biotin-labeled DNA is anchored to SA and the other end is free, it can be captured by Streptavidin-Peroxidase Polymer and appears to be positive on the blot.
The BioA-BspHI-URA3 was incubated with either SA or ddH2O (BioA-BspHI-URA3-SA and BioA-BspHI-URA3-H2O, respectively). For the control, dNTP-BspHI-URA3 and ddH2O were incubated with SA (BspHI-URA3-dNTP-SA and H2O-SA, respectively) and the reaction precipitates were washed by 200 µl 1X PBS 1 to 4 times. The data in Figure 8A shows that BioA-BspHI-URA3-SA still showed positive in the blot after being washed 4 times, and the signal of BioA-BspHI-URA3-H2O was washed away after two washes. There is no signal for dNTP-BspHI-URA3-SA. Therefore, certain proportions of the BioA-BspHI-URA3-SA have at least one end free of SA and the other end bound by SA.
Figure 8B shows the result of BioA-λ DNA and BioC-λ DNA incubated with either SA
or ddH2O (BioA-λ DNA-SA, BioC-λ DNA-SA, BioA-λ DNA-H2O and BioC-λ DNA-H2O, respectively), and that of dNTP-λ DNA and ddH2O incubated with SA (dNTP-λ DNA-SA and H2O-SA, respectively). BioA-λ DNA-SA and BioC-λ DNA-SA were still positive after being washed 4 times and BioA-λ DNA-H2O and BioC-λ DNA-H2O were negative after being washed once. There is no signal for dNTP-λ DNA-SA and H2O-SA. Therefore, certain proportions of the BioA-λ DNA-SA and BioC-λ DNA-SA have at least one end free of SA and the other end bound by SA.
4.1.6 Restriction enzyme digestion of biotin-labeled DNA with SA after 4 washes
To examine whether both ends of biotin-labeled DNA are anchored to SA, restriction enzyme digestion is introduced to cut the DNA into smaller fragments. If both end fragments were anchored to SA, then they will be precipitated down and will not show up in the supernatant, which was subjected to the analysis by electrophoresis.
Figure 9B shows the result of restriction enzyme HincII digestion of BioA-BspHI-URA3-SA, BioA-BspHI-URA3-H2O, dNTP-BspHI-URA3-SA and dNTP-BspHI-URA3-H2O. HincII cut the 3.3 kb DNA fragment into three fragments (1144, 960 and 1237 bps). The1144 bp and 1237 bp DNA fragments contain the original ends and should be anchored to SA (Figure 9A), if they were biotin-labeled. We can see there is a DNA fragment (band J) and a small amount of 1.1-1.2 kb DNA fragment (band K) in the lane of BioA-BspHI-URA3-SA after HincII digestion (Figure 9B). The lane of BioA-BspHI-URA3-H2O, dNTP-BspHI-URA3-SA and dNTP-BspHI-URA3-H2O were negative (Figure 9B). Therefore, it is very likely BioA-BspHI-URA3-SA is filled in with biotin and was able to bind to SA.
Restriction enzyme SmaI cut λ DNA into 19400, 12220, 8271, and 8611 bps DNA fragments, and the 19400 and 8611 bp DNA fragments should be anchored to SA (Figure 10A). The result of the untreated λ DNA digested by SmaI was shown at Figure 10 B and the
band L was the 19400 bps fragment. Figure 10C was the result of BioA-λ DNA-SA, BioC-λ DNA-SA, dNTP-λ DNA-SA after SmaI digestion. It illustrates that there were two DNA fragments on the lane of BioC-λ DNA-SA. One was about 8 kb and the other was greater than 10 kb (band M and N) and the lane of BioA-λ DNA-SA and dNTP-λ DNA-SA were negative after digestion. Therefore, it is very likely BioA-λ DNA-SA and BioC-λ DNA-SA are filled in with biotin and were able to bind to SA.
4.1.7 Fluorescence staining of biotin-labeled DNA with SA
Biotin-labeled DNA with SA was stained with YOYO-1, which allows over a thousand-fold increase in its green fluorescence when bound to dsDNA. The DNA sample was stained with 200 µl 1 µM YOYO-1 at room temperature for 30 minutes, then the stained product was washed with 1 X PBS twice to remove free YOYO-1 and DNA not anchored by SA, and the pellet was resuspended in 1X PBS. 10 µl of the mixture was placed on the slide and observed under fluorescence microscope with 400X magnification.
The results are shown in Figure 11. The data shows that BioA-BspHI-URA3-SA was stained by the green fluorescence after washed, and that of dNTP-BspHI-URA3-SA and H2O-SA were negative after washed.
The staining results of BioA-λ DNA-SA, BioC-λ DNA-SA, dNTP-λ DNA-SA and H2O-SA were shown in Figure 12. The BioC-λ DNA-SA was stained by green fluorescence after washed and the image of BioA-λ DNA-SA shows no significant difference from that of dNTP-λ DNA–SA.