The flowchart in Figure 2.2 details the suppression subtractive hybridisation (SSH) method
FIGURE 2.2 Flowchart detailing the suppression subtractive hybridisation (SSH) method. Steps
are labelled from 2.5.1 to 2.5.4 corresponding to the methods detailed in the text below.
2.5.1 Restriction enzyme digestion
An RsaI digest was set up containing 2 μg amplified DNA, 5 μl 10X RsaI Restriction Buffer (New
England BioLabs, USA), 15 U RsaI and dH2O to 50 μl. The digest was mixed by vortexing and
centrifuged briefly before incubation at 37°C overnight. To confirm that samples in the desired
size range (0.1-2 kb) had been obtained, 5 μl of the digest was run out alongside 5 μl of
undigested DNA and 0.5 μg of 2-Log DNA ladder (New England BioLabs, USA) on a 1% agarose
gel in 0.5X TBE buffer. The gel was stained in ethidium bromide (20 mins), destained in dH2O
(10 mins) and visualised under UV illumination.
Following this, 2 μl of 0.2 M EDTA was added to the remaining digest volume to terminate the
were vortexed thoroughly and centrifuged at maximum speed for 10 mins to separate the
phases. The top aqueous layer was transferred to a fresh 0.5 ml tube and 50 μl chloroform-
isoamyl alcohol (24:1 v/v) was added. The tubes were vortexed, centrifuged and the
supernatant extracted before adding a 0.5 volume of ammonium acetate and 2.5 volumes (of
the total resulting volume) of 95% ethanol. After thorough vortexing, the tubes were
centrifuged at maximum speed for 20 mins to pellet the DNA. The supernatant was discarded
and the pellets overlaid with 200 μl of 80% ethanol. After another 5 minute centrifugation, the
supernatant was discarded and the pellets air-dried for 10 mins. The pellets were then
dissolved in 6.5 μl Milli-Q each and stored at -20°C.
2.5.2 Adaptor ligation
The template for the PCR is created by ligation of adaptor sequences to the DNA to act as
sample-specific priming sites (Figure 2.3).
FIGURE 2.3 Sequence of adaptors 1 and 2 showing the nested primer sites for amplification with PCR Primer 1. (Figure adapted from Clontech PCR-Select™ Bacterial Genome Subtraction Kit Manual, Clontech Laboratories, USA).
The digested tester DNA was diluted 1/2 in Milli-Q and 2 μl of diluted DNA was placed in a 0.5
ml tube together with 4 μl of 5X T4 Ligase Buffer, 800 U of T4 DNA Ligase and 40 pmoles of
Adaptor 1 (5’-CTA ATA CGA CTC ACT ATA GGG CTC GAG CGG CCG CCC GGG CAG GT-3’). The
reaction volume was brought up to 20 μl with Milli-Q and denoted as Tester 1.1. Another 2 μl
of diluted tester was made up in the same way except with Adaptor 1 replaced by Adaptor 2R
(5’-CTA ATA CGA CTC ACT ATA GGG CAG CGT GGT CGC GGC CGA GGT-3’) and this was denoted
as Tester 1.2. 2 μl from each tester tube was mixed in a fresh tube labelled as Tester 1c. All
tubes were centrifuged briefly before incubating overnight at 16°C. The ligation reaction was
stopped by adding 1 μl of 0.2 M EDTA and heating at 72°C for 5 mins. 1 μl of Tester 1c was
diluted in 500 μl of Milli-Q water. Samples were stored at -20°C.
2.5.3 Sample hybridisation
4 μl of digested driver DNA (from section 3.2.2.4 a) was combined with 2 μl of Tester 1.1 and 2
μl 4X SSH Hybridisation Buffer (200 mM HEPES-HCl pH 8.0, 2 M NaCl, 0.8 mM EDTA ph 8.0) in a
tube labelled Hybridisation Sample 1. Another reaction was made up in the same way except
with 2 μl of Tester 1.2 replacing 1.1. This tube was labelled Hybridisation Sample 2. The
samples were overlaid with mineral oil and briefly centrifuged before incubating at 98°C for
1.5 mins followed by a 1.5 hr incubation at 63°C. Another 2 μl of driver DNA was mixed with 2
μl of 2X SSH Hybridisation Buffer, overlaid with mineral oil and denatured by incubating at
98°C for 1.5 mins. A pipette was used to draw up Hybridisation Sample 2, followed by the
freshly denatured driver DNA, leaving a pocket of air in between the two solutions. Both were
then dispensed into the tube containing Hybridisation Sample 1 and mixed by pipetting. After
a brief centrifugation, the reaction was incubated overnight at 63°C. After completing
hybridisation, 200 μl of SSH Dilution Buffer (20 mM HEPES-HCl pH 8.3, 50 mM NaCl, 0.2 mM
EDTA pH 8.0) was added and mixed by pipetting. The sample was heated at 63°C for 7 mins to
2.5.4 PCR Amplification
1 μl of hybridised sample was mixed with 1 μl of Tester 1c in a PCR tube together with 10
pmoles PCR Primer 1 (5’-CTA ATA CGA CTC ACT ATA GGG C-3’), 5 mM dNTP mix, 10X
Thermopol buffer, 1U Taq DNA Polymerase. Milli-Q was added to give a total reaction volume
of 25 μl. The sample was incubated at 72°C for 2 mins to extend the adaptors before
immediately commencing with the PCR. This involved 26 cycles of denaturation at 94°C for 30
seconds, annealing at 66°C for 30 seconds and a 1.5 minute extension at 72°C. After the PCR, a
1 μl volume of product was diluted into 20 μl of Milli-Q. 1 μl of this dilution was placed in a
fresh PCR tube and mixed with 10 pmoles each of Nested Primer 1 (5’-TCG AGC GGC CGC CCG
GGC AGG T-3’ and 5’-ACC TGC CCG G-3’) and Nested Primer 2R (5’-AGC GTG GTC GCG GCC
GAG GT-3’ and 5’-ACC TCG GCC G-3’), 5 mM dNTP mix, 10X Thermopol buffer, 1 U Taq DNA
Polymerase. The reaction volume was made up to 25 μl with Milli-Q and the following PCR
run; 12 cycles of denaturation at 94°C for 30 seconds, annealing at 68°C for 30 seconds, and
extension at 72°C for 1.5 mins. 7 μl of product from each PCR was mixed with loading buffer
and run on a 2% agarose gel. The electrophoresis was conducted in 0.5X TBE buffer at 180 V
for 30 mins before staining in ethidium bromide for 20 mins, destaining in dH2O for 10 mins,
and visualisation under UV transillumination to determine the success of the PCR reactions.