DNA SCIENCE WEEK 1

Objectives:

Restriction Digest and Electrophoresis

 

Introduction:

Plasmids are typically circular double-stranded DNA molecules that replicate within a cell independently of the chromosomal DNA.  They are often used to import recombinant DNA into a host cell for cloning.  More precisely, a DNA fragment that contains a gene of interest is inserted into a cloning vector, or plasmid, and then it can be used to transform a host cell.  The cloning process involves the use of many biological tools.  A restriction enzyme recognizes and cuts DNA at a particular sequence of nucleotides by hydrolyzing the phosphodiester linkage.  Polymerase Chain Reaction (PCR) enables to produce and exponentially amplify a specific gene or gene fragment of interest with appropriate primer design.  For the visual analysis of the restriction digest and PCR product, gel electrophoresis is often employed to separate DNA fragments by their size.

 In the overall experiment, we would extract the lacZ gene from wild type E. Coli (MG1655, GenBank U00096), insert it into a pZE21-GFP vector with kanamycin resistance, and finally transform the E. Coli cell so that it can express lacZ upon the induction of tetracycline promoter.  As the first series of this experiment, cloning vectors were obtained by double digesting pZE21-GFP with KpnI and HindIII.  lacZ gene from wild type E.Coli (MG1655) was then amplified using PCR.  The results were analyzed via gel eletrophoresis.

Materials and Methods:

Restriction Digest:

λ-phase DNA predigest by HindIII was cut by EcoRI enzyme.  pZE21-GFP was digested by three different ways: single digestion by HindIII, single digestion by KpnI, and double digestion by KpnI and HindIII respectively.  For each restriction enzyme used, an optimal buffer was chosen by consulting the endonuclease catalog¹.  In case of double digestion, a single buffer was used by referring to the table of Suggested NEBuffers for Double Digestion at the catalog¹.  Since the selected buffer from the double digest table would not ensure 100% activity of enzymes, higher concentration of vector was added to the mixture.  After selecting appropriate buffers for each sample, reaction solution was prepared by mixing a DNA sample, selected buffer, H₂O, and restriction enzyme to desired DNA concentration into a 1 ml centrifuge tube.   Then sample tubes were spun down to pull down all the liquids.  In order to facilitate digestion, tubes were placed in the 37°C bath for 2 hours.  The reaction was stopped by placing the tube into the 65°C bath for 5 minutes as this would effectively destroy the enzymes. 

 

Polymerase Chain Reaction:

The PCR mix was set up in a 1 ml tube as follows: 0.5 μl of DNA template (MG-1655 and pZE21-GFP), 0.5 μl of forward and reverse primers ordered from IDT (200 nM final concentration of each), 0.4 μl of H₂O, and 22.5 μl of AccuPrime^TM pfx SuperMix (Invitrogen).  Samples were spun down to ensure all liquid is at the bottom.  PCR (Techne, TC-312) was carried out with initial denaturation at 95°C for 5 min; 35 cycles of 95°C for 15 sec (melting), 65°C for 30 sec (annealing), and 68°C for 3 min (elongation) and a final cooling step at 4°C.

 

Gel Electrophoresis

First of all, the agarose gel was heated in the microwave to fully liquefy.  The lane come was placed in the gel tray.  Then the tray was filled with liquefied gel up to 1/3 of its height, ensuring no bubble formation.  Once the gel is solidified, the comb was carefully removed, ensuring no damage to the gel.  1X TAE buffer was poured into the reservoirs so that the gel is submerged with approximately 1 mm depth. 

The loading samples were prepared by mixing the DNA fragments, water, and 6X loading dye properly on the paraffin film so that the final amount of DNA becomes around 150 ng.  Also the loading volume did not exceed 20 μL as it would result in over flooding in the lane.  100 bp and 1 Kbp ladders were also loaded as molecular size markers.  After all sample were loaded, the gel was ran at 100 V for 80 minutes.  The gel was removed from the TAE buffer solution and placed in the Ehidium Bromide staining bath for 10 minutes.  Special caution in handling Ethidium Bromide was required as it is a dangerous mutagen and carcinogen.  After the staining, the gel was transferred to the water bath and rinsed for 20 minutes.  Finally, the gel was placed on the UV light stage and the result was taken as pictures by Sony DSC F707 at wavelength of 302 nm for the visual analysis of restriction digest and PCR products.  The digital image was further processed by MATLAB to ease the quantitative analysis. 

 

Results

            The following DNA samples were loaded on the gel as described before.

Lane	DNA sample	Concentration (ng/ul)	Volume of DNA sample loaded (ul)	Total DNA amount loaded (ng)
4	PCR product of E-Coli		5
5	PCR product of plasmid		5
6	λ-phase/HindIII	500	1.5	750
7	λ-phase/HindIII + EcoRI	30	5	150
8	100 bp ladder	500	1.5	750
9	1 Kbp ladder	500	1.5	750
10	Uncut Vector	13.4	5	67
11	pZE21/HindIII+KpnI	28	5.5	154
12	pZE21/HindIII	6.8	16.7	113.56
13	pZE21/KpnI	6.8	16.7	113.56

Table 1: DNA samples loaded on the gel and their concentration and the amount of samples loaded

 

 

 

 

 

 

 

 

Figure 1: RGB image of the gel (top) and gray image of the gel (bottom)

Figure 2: Image processing with MATLAB helps to identify each band more clearly.

            The number of bands in each lane represented the number of DNA fragments generated by each treatment.  By comparing the distance of each fragment migrated to the relative location of 1 Kb ladder in the gel, the approximate molecular size of each fragment was estimated. 

 

Figure 3: The relationship between the molecular size and the distance migrated in the gel

	# of DNA fragments per λ-phase, E-coli, or pZE21 DNA	Size of each fragment (Kbp)
PCR product of E-Coli	N/A
PCR product of plasmid	1	2.8
λ-phase/HindIII	6	12.9, 7.9, 5.9, 2.0, 1.8, 0.6
λ-phase/HindIII + EcoRI	~7	11.1, 4.7, 4.0, 1.8, 1.7, 1.4, 1.1
Uncut Vector	1	1.7
pZE21/HindIII+KpnI	2	2.2, 0.7
pZE21/HindIII	1	2.9
pZE21/KpnI	1	2.9

Table 2: The estimated number of DNA fragment per λ-phase, E-Coli, or pZE21 as a result of restriction digest or PCR from by the gel electrophoresis analysis

            For restriction digest of pZE21-GFP, the amount of DNA for each kind of fragment and the yield of the digestion was determined by comparing the sample DNA intensity to that of a DNA quantization standard (1Kbp ladder).

Figure 4: 0.5 µg of 1 kb DNA Ladder visualized by ethidium bromide staining on a 0.8% TAE agarose gel (courtesy NEB)[1]

 

	The amount of DNA (ng)	Yield (%)
pZE21/HindIII+KpnI	63 (longer fragment), 80 (shorter)	93
pZE21/HindIII	135	104
pZE21/KpnI	142	110

Table 3: The amount of DNA for each fragment resulted from restriction digest of pZE21-GFP and yield (* yield =)

 

 

Discussion

            By using Vector NTI program (Invitrogen), the actual number of the fragments of their size for each digestion were obtained.  Then they were compared to those we estimated from the gel analysis. 

Figure 5: HindIII sites for λ-phase

Figure 6: HindIII and EcoRI sites for λ-phase

Figure 7: HindIII site for pZE21-GFP

Figure 8: KpnI site for pZE21-GFP

Figure 9: KpnI and HindIII sites for pZE21-GFP

Table 2: The actual number of DNA fragment per λ-phase, E-Coli, or pZE21 as a result of restriction digest or PCR

Figure 10: The comparison between the actual and estimated length of each fragments for HindIII treated λ-phase DNA

            The length of fragments from HindIII treated λ-phase DNA was fairly well estimated by the analysis of the gel electrophoresis.  Only for the longest fragment, the estimation significantly failed.  However, when it was digested additionally with EcoRI, the result from the gel electrophoresis was not instructive as we had too many bends and a couple of them were too vague to identify.  As a result, the number of fragments were actually underestimated for this sample.

Figure 10: The comparison between the actual and estimated length of each fragments for restriction enzyme treated pZE21-GFP

 On the other hand, in case of pZE21, the gel results gave the perfect estimation for both number of fragments and the size of each fragment.  In addition, it was interesting to observe that circular DNA (uncut pZE21) and the linear DNA fragment (either of KpnI or HindIII treated pZE21) moved with different speed even though they have the same length. 

 The yield of the restriction digest was also estimated (Table 3).  Since the optimal reaction buffer cannot be used for the double digest, it did not yield as much digested DNA as the single digest did.  However, the yield estimation through the pixel analysis of the gel image was quite erroneous.  For example, in our analysis, the yield for the single digest was higher than 100%.

Gel electrophoresis indeed was a powerful tool to separate DNA molecules based on the physical characteristics such as size and shape.  We also have seen that some of the quantitative analysis, especially in estimating the size of fragments and the yield of restriction digest, can be done by using the digital image of the gel.  However, at the same time, gel electrophoresis had relatively low quantitative accuracy and limited dynamic range. 

 

References

  1.         Biolabs, N. E. Restriction Endonuclease. http://www.neb.com/nebecomm/default.asp?

 

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