5. ANÀLISI DE DADES
5.5. Anna Poca (8/03/20)
Firstly 100 ng of control DNA (as described in 2.2.1.1) extracted from E. coli K12, C. difficile or A.naeslundii were incubated with Nt.BstNBI in the following reaction scaled down from a generic restriction enzyme protocol. Input DNA 100 ng, 1 µl 10x buffer (NEB), 1U Nt. BstNBI enzyme (NEB) and water to a total volume of 10 µl.
The reaction was incubated at 55oC for 1 hour. The enzyme was then de activated at 80oC for 20 minutes. The DNA was cleaned up using isopropanol precipitation. Briefly 5 µl 7.5M NH4OAc (Sigma-Aldrich) and 30 µl 100% isopropanol (Sigma-Aldrich) was added to the samples and
incubated at room temperature for 10 minutes. This was then centrifuged at 4oC for 10 minutes at 21000xg. The pellet was then washed with 70% ethanol and centrifuged as before. The ethanol was removed and the sample allowed to air-dry for 15 minutes before being resuspended in 15 µl
48 of molecular grade water. The sample was then quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and stored at -20oC.
2.2.3.2. MDA Reaction with Nicked DNA
DNA was nicked and cleaned up as described in 2.2.3.1 then four ɸ29 MDA reactions were set up for each isolate,
1. Non-nicked with primer control 2. Non-nicked without primer 3. Nicked DNA with primer 4. Nicked DNA no primers.
The ɸ29 enzyme for all nicking work was supplied by NEB, with specific 10x ɸ 29 DNA
polymerase reaction buffer. Samples which included primers were denatured prior to addition to the reaction mix below. 0.5 ng of DNA was added to the reactions. 5 µl of ɸ29 10x buffer, BSA to 0.2 µg/ µl, 200 µM dNTPs, 10U ɸ 29, 250ng Endonuclease resistant random hexamers (where needed) and water to a total volume of 50 µl
The reactions were then incubated at 30oC for 6 hours and DNA was quantified at 1, 2, 3, 4, 5 and 6 hours, and inactivated at 65oC for 10 minutes.
2.2.3.3. Co-nicking and MDA Amplification
The nicking and amplification reactions were combined without a clean-up stage. 0.5 ng of DNA was added to the above nicking reaction (2.2.3.1) and incubated for 30 minutes at 55oC, then ramped down 30oC before addition of the MDA reaction mix (2.2.3.2). The same four
combinations of reactions were used as previously, with the samples including pri mers denatured before addition to ɸ29 MDA mix.
2.2.3.4. Use of T4 Gene 32 Protein for Amplification Stabilisation
T4 Gene 32 Protein (NEB) is a single stranded binding protein (SSBP), which stabilises single stranded DNA, preventing re-annealing. This was previously reported96 to increase DNA yields during amplification at a level of 2.5 µg/100 µl. The T4 Gene 32 Protein was supplied at a concentration of 10 mg/ml, in 50 µl reactions 1.5 µg was added (1.5 µl 1:10 dilution).
49
2.2.4. Combining Displacement Enzymes
Other polymerases with strong stand displacement activity were combined with ɸ29 to investigate the impact on extension from nicks. For assessment of the potential use of enzyme combinations with ɸ29, E. coli K12 cultured control DNA was extracted as described in 2.2.1.1 and nicked as described in 2.2.3.1.
2.2.4.1. Klenow Fragment (3′→5′ exo-)
Klenow fragment has been previously used to produced DNA from nicks 97 and so was investigated in isolation and in combination with ɸ29. The following reaction mix combinations were trialled
1. Klenow Fragment with nicked DNA and random primers 2. Klenow Fragment with nicked DNA
3. Klenow Fragment with nicked DNA and SSBP
4. Klenow Fragment and ɸ29 with nicked DNA and random primers 5. Klenow Fragment and ɸ29 with nicked DNA
6. Klenow Fragment and ɸ29 with nicked DNA and SSBP
Klenow Fragment (NEB) was supplied with 10 x NEB buffer 2 (NEB). 2.5 µl was added to the reaction along with 2.5 µl 10 x ɸ29 DNA Polymerase Reaction Buffer (NEB) to create a
combination reaction buffer. To this buffer mix 0.2 µg/ µl of Bovine Serum Albumin (BSA), 200 µM dNTPs and 10 U of Klenow fragment were added along with the required combination of the following components:
250ng Endonuclease resistant random hexamers
1.5 µg T4 Gene 32 Protein (NEB)
10U of ɸ29
10 ng nicked DNA was added and reactions made up to a total volume of 50 µl with water. If samples had primers added DNA was denatured by heating to 95oC for 2 minutes and held on ice for one minute before addition to the reaction mix. All reactions were then incubated at 30oC for six hours.
50 Deep VentR has known strand displacement activity, but is normally used for thermo cycling production of DNA, where there are high levels of secondary structure. Initially a thermocycling reaction was set up using the E. coli K12 primers described in 2.2.1.6
A 50 μl reactionwas set up, usingDeep VentR DNA Polymerase (NEB). The mix consisted of
5
μl ThermoPolReaction Buffer (10X), 1 μl dNTP mix, 1.25 µl 20µM forward and reverse primer, 10 ng denatured DNA, 0.5 μl Deep Vent DNA Polymerase and water to 50 µl. The cycling condition were as follows, 95oC 5 minutes, and 30 cycles 94oC 30 seconds, 59oC 30 seconds, 72oC 30 seconds, hold at 72oC 10 minutes, and a final hold at 4oC.To assess the potential for isothermal amplification the same reaction mix was used, with 250 ng endonuclease resistant random hexamers used in place of the specific primers. 10 ng
denatured DNA was added to the reaction which was held at 30oC for 1 minute, and either held at 30oC for six hours or the temperature was ramped up to 40oC, 50oC, 60oC, 70oC or 80oC for six hours.
To test for ability to extend from nicks, 10 ng of nicked DNA was added to the reaction mix without primers, with or without the addition of 1.5 µg T4 Gene 32 Protein. This was then incubated at 70oC or 80oC for six hours.
2.2.4.3. Bst DNA Polymerase, Large Fragment
50 µl reactions using Bst DNA Polymerase, Large Fragment and 10X ThermoPol Reaction Buffer (NEB). ). 5 μl ThermoPolReaction Buffer (10X), 3 µl MgSO4 (100 mM), 2 μl dNTP mix, 10 ng nicked DNA, 10 U Bst DNA polymerase and water to 50 µl. Where necessary 1.5 µg of T4 Gene 32 Protein (NEB) was added. The sample was then held for six hours at 30oC, 40oC, 50oC, 60oC, or 70oC.
When combined with ɸ29 2.5 µl ThermoPolReaction Buffer (10X) and 2.5 µl 10 x ɸ29 DNA Polymerase Reaction Buffer was used in the above reaction mix. For the primer control 250 ng endonuclease resistant random hexamers were added. Before addition of the ɸ29 to the mixture (including Bst) was held at 50oC, 60oC, or 70oC for one hour before cooling to 30oC and the ɸ29 (10U) added.
2.2.5. Amplification of DNA using DNA tagging
Addition of nucleic acid tags to the 3’ end of nucleic acids has the potential to stabilise the nucleic acid and produce more full length transcripts by allowing replication to initiate upstream
51 of the start of the target DNA. By limiting the start of replication to a single sight amplification bias and over amplification of regions will be limited. Addition of tags also give s the potential to add barcodes, allowing initial abundance in samples to be calculated.