Activity 1.

PCR Amplification of 16S rDNA for Bacterial Identification

Bacterial 16S rDNA sequencing is a molecular method used to determine the genus and species of an unknown bacterial isolate. 16S rDNA is the gene encoding for prokaryotic 16S
rRNA, with a length of about 1500 bp. It is found in the genomes of all prokaryotes, and is made up of multiple conserved and variable regions (Zheng et al., 2021). The conserved
region is shared by all bacteria, and there are no significant differences between them. While the variable region varies between bacteria and is unique to each genus or species of
bacteria (Reller et al., 2007).

The use of PCR to amplify 16S rDNA employs universal PCR primers in the conserved region, which can identify all kinds of bacteria present in the sample and PCR-specific primers
based on the variable region, which can identify bacteria up to species and strain level (Sontakke et al., 2009). Bacterial 16S rDNA has nine variable regions labeled V1-V9 (Jumpstart
Consortium Human Microbiome Project Data Generation Working Group, 2012).

In general, if two strains have equal or greater than 99% homology in full length 16S rDNA sequence, they are considered the same bacteria. If homology is 97%-98%, it is considered
to be a different species of the same genus. If the percentage is less than 95%, it is considered to be a different genus (Zheng et al., 2021).

Objectives

1. Describe how to amplify 16S rDNA from bacterial samples using PCR.
2. Explain principle of using 16S rDNA for bacterial identification.
A. Bacterial DNA Extraction using CTAB Method

The ionic detergent cetyltrimethylammonium bromide (CTAB) is used to disrupt membranes, and a chloroform-isoamyl alcohol mixture separates contaminants into the
organic phase and nucleic acid into the aqueous phase is the conventional method for extracting DNA (Table 1).

Materials

1. Bacterial culture in broth media

2. 1. 5 mL Microcentrifuge tubes or Eppendorf tubes
3. Microcentrifuge tube racks
4. Refrigerated centrifuge
5. Micropipettes
6. Pipette tips
7. 50 mL conical tubes
8. Conical tube rack
9. Ice bucket
10. Gauze

2
Table 1. CTAB method for DNA extraction

Step Procedure
1. Collect Cells Centrifuge 1.5 mL of bacterial broth culture using microcentrifuge tubes at 12,000 × g for 2 minutes. Discard the supernatant.

2. Resuspend Cells Add 700 μL Lysis buffer into the cell pellet and resuspend cells by pipetting in and out.
Then add 10 μL RNAse A (10 mg/mL), incubate at 37°C for 1 hour. This will degrade RNA that will contaminate the DNA extract.
3. Lyse Cells Add 10 μL Proteinase K (20 mg/mL) to cell suspension, vortex or pipet to mix. Incubate at 50 °C overnight. This will lyse the bacterial cell
wall, releasing the cytoplasmic contents.
4. Separation Add 700 μL of Phenol/Chloroform/Isoamyl alcohol (25:24:1) and mix well by inverting the tube. This will separate the lipids and cellular
debris containing proteins into the organic phase (bottom part of the solution), and the DNA into the upper aqueous phase.
Centrifuge at 13,000 rpm for 10 minutes. Pipet aqueous phase and transfer into a fresh tube. Then repeat step 4 twice.
5. Protein Denaturation Add 700 μL chloroform and mix well. This will enhance the denaturation of the remaining proteins, phenols, and lipids in the solution.
Chloroform also enhances the separation of the organic phase and aqueous phase, which contains the DNA.
Centrifuge at 13,000 rpm for 10 minutes. Pipet the aqueous phase and transfer into a new tube. Repeat for one time.
6. DNA Precipitation Add one tenth volume of 3M sodium acetate (NaOAc). Then add two volumes of 100% ethanol (EtOH). DNA will start to precipitate
immediately after mixing. Incubate DNA at -70°C for one day to further enhance the precipitation.
Centrifuge at 13,000 rpm for 30 minutes at 4°C. Discard supernatant.
7. Washing Wash with 70% EtOH. Centrifuge at 13,000 rpm for 10 minutes. Discard supernatant. Remove remaining EtOH using pipet. Then air dry at
room temperature.
8. Elution Add 200 μL of TE buffer or nuclease-free water. Tris-EDTA (TE) buffer dissolves the DNA and protects it from degradation by DNase or
RNase. Store gDNA in 4°C, -20°C or -80°C.

3
B. Preparation of PCR Master Mix

PCR master mix is usually prepared separately in a 1.5 microcentrifuge tube before adding into the PCR tube containing the primers and DNA template.

Materials

1. Bacterial genomic DNA

2. KOD-Plus-Neo POL (Taq DNA Polymerase)
3. 10X KOD-Plus-Neo Buffer
4. dNTP mix
5. MgSO4
6. 16S rDNA primers: 27F (F-5′-AGAGTTTGATCNTGGCTCAG-3′) and ipRSR2 (R-5′-AAGGAGGTGATCCANCCGC-3′)
7. Sterile distilled H2O or nuclease-free H2O
8. PCR tubes
9. Sterile 1.5 mL microcentrifuge tubes
10. Micropipettes
11. Sterile pipette tips
12. Ice bucket

4
Methods

1. Use sterile pipette tips, microcentrifuge tubes, and PCR tubes in preparing the PCR master mix.
2. Use fresh gloves. Disinfect with 70% EtOH.
3. Work in a PCR hood and put all PCR reagents in an ice bucket. Disinfect the bench with 70% EtOH. Dust can be a source of contaminants for PCR reagents and DNA
sample.
4. Compute the volumes required for each of the PCR components in a 20 μL vol/rxn for 21 reactions (Table 2).

C1V1=C2V2
C1= Stock concentration C2= Final concentration
V1= Volume required from stock solution V2= Volume/rxn

Table 2. PCR Master Mix

Components Stock Concentration Final Concentration 20 μL Vol/rxn (V2) Total Vol (20+1 rxns)
(C1) (C2) (μL) (μL)
1. 10X KOD-Plus-Neo Buffer 10X 1X-2.5X (V1) 5 105
2. MgSO4 25 mM 1.5-2 mM 1 21
3. dNTP mix 2 mM 50-500 μM 1 21
4. Forward primer 10 μM 0.1-0.5 μM 0.5 (10.5)**
5. Reverse primer 10 μM 0.1-0.5 μM 0.5 (10.5)**
6. KOD-Plus-Neo POL 5.0 U/μL 0.25-2.5 U/μL 1 21
7. DNA (50-500 ng/ μL)* (50-500 ng/ μL)* 2 (42)**
8. ds H2O (nuclease-free H2O) - - 9 189
9. Total volume - - 20 420

* DNA concentration may vary

** Do not include in the PCR master mix

5
5. After determining the volume required for each PCR reagent per reaction (20 μL), multiply with the number of reactions to be made (additional volume for one reaction
is usually incorporated in the total volume to cover possible inaccuracy in pipetting along the preparation).
6. Use micropipette to aspirate the required volume of each PCR component (KOD-Plus-Neo POL, PCR Buffer, MgSO4, dNTP mix, ds H2O) and put in a 1.5 mL
microcentrifuge tube. After adding all the components, mix well by pipetting in and out or by vortexing.
7. In a PCR tube, add separately the forward primer (0.5 μL), followed by the reverse primer (0.5 μL), and lastly add the DNA template (2 μL).
8. Add the PCR master mix (17 μL) on each PCR tube and mix by pipetting in and out.
9. This is now ready for PCR.

6
C. Thermal Cycling Conditions

PCR thermal cycling profile commonly includes pre-denaturation, denaturation, annealing, extension, and final extension steps. Twenty to 35 cycles of denaturation,
annealing, and extension can produce a million copies of the target DNA sequence for 2.5 hours or less depending on the cycle profile and efficiency of thermal cycler.
Thermal cycling conditions like the PCR mix should be optimized. Annealing temperature where primers attached to the target genes or DNA sequence has the most
influence in a PCR profile (Table 3).

Table 3. PCR Cycle Profile

Steps Temperature (°C) Duration Cycle(s)

1. Pre-denaturation 94 2 min 1
2. Denaturation 95 10 s
3. Annealing 60 10 s 35
4. Extension 68 60 s
5. Final Extension 68 60 s 1

7
Questions for Research

1. Why do you think ice bucket is needed during the preparation of PCR reactions?
2. What is the purpose of using a thermostable DNA polymerase for PCR?
3. Why optimization of annealing temperature is important in PCR?

References

1. 16S rDNA Amplification. Retrieved from https://www.the-odin.com/16s-rdna-amplification/

2. DNA Extraction by Phenol Chloroform. Retrieved from http://www.abnova.com
3. Jumpstart Consortium Human Microbiome Project Data Generation Working Group (2012) Evaluation of 16S rDNA-Based Community Profiling for Human Microbiome
Research. PLoS ONE, 7, e39315
4. Reller BL, Weinstein MP, Petti CA (2007) Detection and Identification of Microorganisms by Gene Amplification and Sequencing. Clin Infect Dis, 44, 1108-1114
5. Sontakke S, Cadenas MB, Maggi RG, Diniz PPVP, Breitschwerdt EB (2009) Use of Broad Range 16S rDNA PCR in Clinical Microbiology. J Microbiol Methods, 76, 217-225
6. Tamari F, Hinkley CS, Ramprashad N (2013) A Comparison of DNA Extraction Methods using Petunia hybrida Tissues. J Biomol Tech. 24(3): 113–118
7. Zheng J, Zhu J, Chen B, Chen LX, Gao T, Chen XP, Li FY (2021) Application of 16S rDNA Sequencing Technology in the Analysis of Pathogenic Bacteria in Sputum of Severe
Pneumonia. Adv Microbiol, 11, 157-164