PCR For 16S rRNA Sequence Analysis

PCR For 16S rRNA Sequence Analysis:-

Principle:-

> Polymerase chain reaction (PCR) is molecular technique used to amplify specific regions of DNA for applications such as sequencing and genetic analysis. 

>Typically, there is a limited amount of DNA in the sample to study and amplification is required. 

> PCR is carried out in a test tube with the DNA template, primers specific for the region that is desired, DNA polymerase, and reagents that stabilize the reaction. 

> Once the reaction is put together, it will go into a thermocycler (PCR machine) that will create the conditions for DNA replication to occur. 

> Each round of PCR requires three steps, denaturation, annealing, and elongation, each of which doubles the amount of DNA template present in the reaction. 

> By repeating this process multiple times, usually 30, this will amplify the DNA exponentially.

PCR bead method:-

Materials:-

i. 27F primer (20uM stock)

ii. 1492R primer (20uM stock)

iii. GE Illustra PuReTaq Ready to go PCR bead and tube

iv. Sterile nuclease-free deionized water (molecular grade)

v.  T-Streak plate with bacterial isolate

vi. Micropipettors and tips (P10, P100)

Procedures:-

> Obtain PCR bead tubes, which contain Taq polymerase (heat resistant enzyme) and other necessary reagents. 

> Using a sharpie, label the top of the tubes with PCR reaction number assigned in class.  

> Make sure not to accidentally rub this off when handling the tube and double check when you put the tube into the PCR machine that your labeling is still visible.

> Add 25 μL of Master mix (contains molecular grade water + 16S rRNA primers) into the PCR bead tube. The bead will start to dissolve and slightly effervesce.

> As you dispense the Master mix, insert the micropipette tip into the mix so that you actually see the small volume go directly into the mix.

> Using a micropipette tip, carefully touch the colony on the streak plate. A small, visible dab of cells that barely fill the very end of the pipette tip will provide enough DNA template for the reaction.

> Dip pipette tip into reaction mix and gently swirl for 5-10 seconds to dislodge cells. Cap the tubes. Avoid forming bubbles.

> Transfer tubes to thermal cycler.

> Select appropriate program† to start cycling (about 2 hours).

> Once cycling is complete, remove tubes and incubate on ice. Follow your instructor’s instructions about storage, and follow up protocols to quality test the PCR products and prepare them for sequencing.

16S rRNA Primers:-

i. Forward Primer (27F):-

5’ – AGA GTT TGA TCC TGG CTC AG – 3’

ii. Reverse Primer (1492R):-

5’ – ACG GCT ACC TTG TTA CGA CTT – 3’

PCR Cycle Protocol:-

i. 94℃ for 10 min.

ii. 94℃ 30 sec – Denaturation step.

iii. 58℃ 30 sec  - Annealing step.

iv. 72℃ 1 min 50 sec (1 min per kb of DNA template) – Elongation step.

v.  Repeat Steps 2-4 30X.

vi. 72℃ for 10min – Final extension step.