LAB 5: IDENTIFICATION OF PATHOGENIC BACTERIA BY PCR AMPLIFICATION OF THE 16s rRNA GENE & RESTRICTION FRAGMENT

1. LAB 5: IDENTIFICATION OF PATHOGENIC BACTERIA BY PCR AMPLIFICATION OF THE 16s rRNA GENE & RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP)

2. OBJECTIVES • Describe process & principles of PCR technique • Describe how the 16 S rRNA can be used in bacterial identification • Use PCR amplification technique to amplify the 16S rRNA of selected bacteria • Use restriction endonucleases to digest 16S rRNA amplicons and generate banding patterns that allow the identification of selected bacteria

3. Polymerase Chain Reaction Use a DNA polymerase to amplify a specific sequence exponentially Requires: • Target (template) DNA to be amplify • Primer to anneal to the beginning of selected area in DNA template (forward primer) • Primer to anneal to the end of selected area in DNA template (reverse primer) • DNA polymerase, nucleotides, proper conditions for DNApol activity reverse primer 5’ 3’ 3’ 5’ target sequence forward primer

4. 5’ 3’ 3’ 5’ reverse primer 5’ 3’ forward primer 3’ 5’ 1) Denature template DNA, add primers , allow to anneal 2) Allow DNApol to synthesize copy of target sequence 5’ 3’ 3’ 5’ 3) denature again , allow primers to bind to target DNA again

5. 5’ 3’ 3’ 5’ 4) Allow the DNApol to synthesize again, the result is now 3 new copies of the original target 5) As denaturing, annealing, synthesis cycles are repeated, the number of copies will double (exp. increase)

6. DNA pol used in PCR • In principle any polymerase would do. DNA pol III was used in the first PCRs. Heat resistant pol preferred Why? Taq polymerase (Thermus acuaticus) stable at 95◦C, optimun synthesis at 72 ◦C, not proof-reading Pfu polymerase (Pyrococcus furius) stable at 100◦C, synthesis at 70-75 ◦C, proof-reading A vast array of Thermostable DNApol capable of amplifying different sizes fragments are now commercially available

7. Primers for PCR Ref. Protocol for PCR with Pfu DNA Polymerasewww.fermentas.com/techinfo/pcr/pcrprotocolpfu.htm 20-30 nt long GC content between 40-60% in even distribution More that 3 G or C nt at 3’ end can result on unspecific annealing Check primer sequences to avoid the following: • sites of complementarity between primers and template • self complementarity (producing hairpin structures) • complementarity between primers (produces primer dimers) • Degenerate primers must have at least 3 conservative nucleotides at the 3’ end • Melting temp. of forward & reverse primers must not differ more than 5 C

8. Calculating annealing temperature of a primer: Tm = 4(G + C) + 2(A + T) where G,C,A,T are the number of respective nucleotides This formula can be used for primeas that are no longer than 25 nt long Nowadays, some specialized computer programs can do all calculations and determinations for proposed primers e.g. Gene runner

9. Other key factors for PCR Proper MgCl2 or MgSO4 concentration • Each polymerase has a preferred source & appropriated [ Mg] concentration. This must be calculated for each experiment • Mg2+ ions form complexes with the nucleotides, primers, and templates • Too little Mg will result in low PCR product & increase of non-specific products dNTPs: • must be present in equal amounts, standard concentration is 200 μM

10. Proper temperatures & cycling; • Initial denaturation : aim to completely denature template DNA. 95 C & 1-3 min (less that 50% GC) to 10 min (GC rich template) • Primer Annealing: Allow primers to base pair with the proper sequence on template. Temp. 5C lower that the melting temp of the primer template duplex. Between 0.5-2 min. If non-specific products are produced increase annealing step Denaturation Step: at end of each round of replication, templates are denature to allow new primer annealing

11. Extension: ~ 2 minutes for every Kb to be synthesized • Number of cycles: Depends on amount of template & expected yield, 25-35 cycles lower # of cycles reduces background of non-specific products. Final Extension: 5-15 min allowed to fill-in the protruding ends of newly synthesize PCR products

12. 16s rRNA PCR-RFLP • 16S ribosomal RNA, essential structural prokaryotic ribosomes • gene for 16S rRNA highly conserved in all bacteria, 4-6 copies • 16s rRNA gene has regions of stable sequence common to all bacteria & several regions of variable sequences. Useful for study evolutionary relationships and also for ID • Variable regions or polymorphisms, have unique specie’s specific sequences. Restriction enzymes can be used to cleave the 16S rRNA gene into several small fragments. • Use of enzymes that cut in variable regions of the 16S rRNA will produce a species’ characteristic number & sizes of restriction fragments, separable by G.E. The banding pattern unique to each species of bacteria produced in this manner is called : restriction fragment length polymorphism (RFLPs). • Usable a diagnostic tool to identify bacteria at the species level.

14. PCR OF BACTERIAL16s rRNA GENE Forward primer called 68F: 5’ TNANACATGCAAGTCGAKCG 3’ (Mullins et.al. 1995) Reverse primer called 1406R: 5’ ACGGGCGGTGTGTRC 3’ (Amann et al. 1995) Where Y= T or C, R= A or G, D= A, G, or T and N= A, C, T, or G