Biology 475

Molecular Biology

PCR for 16S rRNA and Cloning

National Science Foundation

Western Oregon University

Yellowstone National Park

PCR stands for "polymerase chain reaction." PCR is the basis for rapid and extremely sensitive genetic, medical, and criminal testing. It enables one to detect as little as one target sequence (whole genes, gene fragments, or non-coding) and, from this, amplify billions of identical copies, a visible band of DNA on a gel. PCR works by forcing DNA replication at sites defined by man-made primers. As should be evident, you need to know something about your target because you have to construct the primers.

Another important feature of PCR is the use of a thermal-stable DNA polymerase (Taq polymerase). During replication in vivo, DNA unwinding is facilitated by dozens of cellular proteins. In contrast, PCR utilizes heat (92-95 degrees C) to denature the template DNA. Mesophilic polymerases denature at these temperatures. Prior to 1980, scientists used animal enzymes to replicate small amounts of PCR product after boiling DNA, adding DNA Pol, and allowing extension. The practicality of this approach was extremely low (detectable products require 20+ repetitions). In 1980, Kerry Mullis thought to use a thermal-stable polymerase and PCR became a real-time possibility. Mullis isolated Taq from Thermus aquaticus, a bacterium that thrives in 70 degree C hot springs. Thermus was originally isolated from Yellowstone in 1969 by a microbial ecology professor named Thomas Brock. Mullis' contribution to PCR technology earned him the Nobel Prize in Chemistry and annual reagent revenues totaling 750 million a year. Even so, Mullis has received criticism over the years. Many scientists believe that the Nobel should not have been awarded to Mullis because others had clearly developed the basic concept for the method. Also, Yellowstone has never prospered directly from Taq profits and this has resulted in court-contended attempts by the park to sign exclusive "bioprospecting" deals with biotechnology companies. Despite everything, Taq polymerase represents a tool with amazing applied uses - despite originating from basic research driven solely by curiosity. Remember that the next time you hear someone criticizing studying something that sounds trite... or when you hear about threats to flat-line basic research funding at the federal level.

Troubleshooting PCR - False Negatives
Despite how easy PCR sounds, it can be difficult for many reasons. Amplifying DNA from environmental samples is tricky because many habitats have metals, acids, or bases that, even in extremely trace amounts, inhibit Taq. Also, too much starting template DNA can inhibit PCR. PCR troubleshooting methods that address both of these problems entail (1) amplifying samples in different buffers with varying acids or bases; and (2) amplifying a dilution series of your starting template.

Troubleshooting PCR - False Positives
On the flip side, false positives are a serious threat because optimized PCR is SOOOOO sensitive. Once a lab becomes established, it is imperative that areas where template DNA is initially purified and where PCR reactions are set up remain free of contaminating product. We have designated chemical stocks, water, equipment, and space to a "P-Free" zone (where P stands for plasmid and PCR product).

Our PCR Reactions
All PCR reactions contain five things in addition to background water. Our target gene is a common fingerprinting gene - the 16S ribosomal RNA gene - used to compare and identify bacteria.

Template DNA: genomic DNA extracted and purified from the red layer.
Buffer: We use Epicenter's MasterAmp kit.
Forward Primer: man-made primer that binds 5'-upstream portion of target.
Reverse Primer: man-made primer that binds 3'-downstream portion of target.
Taq Pol: thermal stable polymerase, now harvested from recombinant E. coli.

Our PCR Cycling Conditions
All PCR cycles involve three steps.

Denaturing: 94 degrees C for 1 minute.

Annealing: 1 minute for 60 degrees C. Depends on primer composition. Roughly = 3(# G+C) + 2(# A+T).

Extension: 3 minutes at 72 degrees C. Depends on the predicted length of the product. Roughly = 1 minute per 500-1000 bases.

Number of Cycles: 35 repetitions. This is variable and somewhat contentious. The biggest argument against MANY cycles: Taq polymerase errors that yield mutations.

Procedures
In this exercise, each person will set up ten reactions using a single template sample. Specifically, you will amplify starting template with two different primer sets, each using six buffers (i.e. 5X2 = 10).

Standard PCR operating procedures - close all lids as you work… no open tubes!

Make sure everything is thawed, mixed, and on ice before beginning.

Label 10 small centrifuge tubes with your sample, the buffer, and the primer used.

Buffers will be: B, D, H, J, L
Primers will be: 16S set (F/R) and Red set (F/R)

Using a different tip each time, add 25 ul of each buffer to each appropriate tube (B into B, D into D, and so on).

Move the rack of sample buffers to the side and set up two new tubes. These will contain "cocktails" that has enough of everything else - one cocktail for 16S primers and one for Red primers. Each will be distributed into the tubes you just set up. Because of "slop factor," your cocktails will each contain enough stuff for six tubes. Mix, in the following order:

6X Cocktail (make two of these one for each primer set)
131 ul water
6 ul forward primer
6 ul reverse primer
1.2 ul Taq Polymerase
6 ul DNA (always last - after everything else is closed and put away)

Gently mix your cocktail and, using a new tip each time, add 25 ul to each of your buffer tubes.

Place tubes on ice and carry to the PCR machine. Load and turn on the machine. Select File = 45 (enter) and then start. This program has already been written into the machine to the specifications listed above. The machine will take three and a half hours.

Primer Homework
In this hands-on assignment, you will determine where each of the following primers binds in terms of our favorite bacterium, Roseiflexus. In this exercise, you will be given a GenBank file that provides 16S rRNA sequence information about Roseiflexus, a red filamentous bacterium from hot springs in Japan. Using your eyes and logic, determine where each of these primers binds and how long the predicted PCR product will be. Note: this is NOT as easy as it looks and, unfortunately, BLAST no longer works on such short input queries!!!

16S Forward: 5'-GCGGATCCGCGGCCGCTGCAGATGTTGATCCTGGCTCAG-3'

16S Reverse: 5'-GGCTCGAGCGGCCGCCCGGGTTACCTTGTTACGACTT-3'

Red Forward: 5'-GTGGCGNACGGCTGAGG-3'

Red Reverse: 5'-GACGGNCCCTCGNAGGC-3'

N = ANY base