Today, the world of genetic engineering and molecular biology is an extraordinarily high-tech world of dazzling equipment. Machines such as automated DNA sequencers, automated thermal cyclers, automated DNA synthesizers, and powerful computer hardware and software that can synthesize and interpret the mind-boggling array of data that are commonplace even in relatively small research labs.
Polymerase Chain Reaction (PCR)
The polymerase chain reaction is the backbone of most labs that manipulate and study DNA. The reaction is basically a Xerox machine for DNA. We first design PCR primers that complement the ends of the DNA target sequence we want to amplify and then make several million copies of that sequence. The result is enough DNA of the specific target sequence to be analyzed on a DNA sequencer or to be "seen" on a gel. The reaction is based on the enzymatic amplification, or copying, of a specific DNA fragment, or target sequence, flanked by oligonucleotide primers that hybridize to opposite strands of the target sequence. Heat denatures the original double stranded target DNA into two complementary single strands (see DNA structure here), allowing the primers to anneal to their complementary sequences. Extension of the annealed primers with a DNA polymerase that remains functional at high temperatures (such as Taq DNA polymerase from the hotspring bacterium Thermus aquaticus) results in the amplification of the target DNA sequence. Each cycle of denaturing, annealing, and extension doubles the previous amount of target DNA that is present. Several million fold copies of the target DNA sequence can be made in just a few hours.
Here is our PCR protocol for genomic soybean DNA amplification using primers to SSR flanking regions (per reaction):
1. 30 ng genomic soybean DNA
50 mM KCl
10 mM Tris-HCl (pH 9.0 @ 25° C)
0.1% Triton X-100
3. 1.5 mM MgCl2
4. 0.15 mM for each of the NTPs (A,T,G,C)
5. 1 unit Taq DNA polymerase
1. 2 minutes @ 95º C
2. 33 cycles of:
Denaturation: 92º C
Annealing: 47º C (46º also works quite well if 47º produces poor results)
Extension: 68º C
Use equal times for denaturation, annealing, and extension. Time depends on PCR machine, reaction volume, etc.