In this lab you will perform site-directed mutagenesis using the QuickChange mutagenesis kit (Stratagene). You will learn how polymerases work and how to amplify DNA using polymerase chain reaction (PCR).
4.2 Mini Project Flowchart
The bolded block in the flowchart below highlights the role of the current experiment in the mini project.
4.3 Review of Nucleic Acid Structure
Before you learn how polymerases work and the requirements of a successful PCR amplification you must review a few things about nucleic acid structure. Nucleic acids are polymers of nucleotides. The nucleotides are held together by phosphodiester bonds in the nucleic acid. Figure 4.1 shows the structure of a nucleotide. Nucleotides are made of a sugar moiety: ribose (RNA) or deoxyribose (DNA); a heterocyclic aromatic moiety: the nucleobase (A, C, G, T or U) and a phosphate group. When we refer to functional groups within the sugar moiety we use the apostrophe symbol; for example the second hydroxyl group on the ribose in the nucleotide is referred to as the 2'-OH group.
As we mentioned earlier, nucleotides are held together by the phosphodiester bond in the nucleic acid chain. The phosphodiester bond is made out of two phosphate ester bonds: each is formed between a OH group of the ribose or deoxyribose and a OH group of the phosphate. Since phosphoric acid is a moderately strong acid, the phosphodiester bond deprotonates under physiological conditions giving nucleic acids a negative charge (Fig. 4.2).
Nucleic acid chains have polarity just like protein chains. Nucleic acid polymers start with the 5’ phosphate of the first nucleotide and end with the 3'-OH group of the last nucleotide in the chain. Therefore the chain of the nucleic acid has a precise direction; it goes from 5' to 3' direction end just like protein chains go from the N-terminus to the C-terminus. In a double-stranded nucleic acid like DNA (our genetic material), one DNA polymer goes 5’ to 3’ and is called the top strand and the other goes from 3' to 5' direction is called the bottom strand. These two DNA strands are complementary to each other: adenine (A) against thymine (T) and cytosine (C) against guanine (G) to ensure proper Watson-Crick base pairing between the strands. In other words the two DNA strands in this double-stranded DNA (dsDNA) are complement of each other. Complement means that the two strands are complementary to each other and the chain direction is opposite: the top strand goes 5’ to 3’ whereas the bottom strand goes 3’ to 5’.
5’ AGGCCATTGGA 3’
3’ TCCGGTAACCT 5’
4.4 How do Polymerases Work?
Polymerases synthesize nucleic acids using a nucleic acid template. The sequence of the newly synthesized nucleic acid will be complement of the template sequence. During nucleic acid synthesis, the 3'-OH group of the growing nucleotide chain acts as a nucleophile to attack the phosphorous of the incoming nucleotide, a pyrophosphate (PPi) leaves and the phosphodiester bond forms. This means that the newly synthesized DNA chain grows in the 5’ to 3’ direction (Fig. 4.3).
To properly position the nucleophile for attack divalent metal ions (usually Mg2+) are necessary for successful DNA synthesis.
Polymerase mechanism in a nutshell:
– Polymerases synthesize DNA using a template that is the complement of the newly synthesized DNA.
– To synthesize DNA, polymerases require an OH group to act as a nucleophile. This OH comes from the growing nucleic acid chain. Recognize that the reaction is a nucleophilic substitution.
– The leaving group is pyrophosphate (PPi), which is a high-energy molecule that splits into two inorganic phosphates. This reaction is catalyzed by the enzyme inorganic phosphatase in vivo.
– Polymerization is energetically favorable, because two high energy anhydride bonds are broken (one in the incoming nucleotide triphosphate and the other in pyrophosphate) and one stable bond forms (ester bond connecting the nucleotides).
– To initiate DNA synthesis, DNA polymerases require a primer to provide the required OH group as nucleophile.
– Polymerases travel from 3' to 5' direction on the bottom strand of the dsDNA template while they synthesize the growing chain in the 5’ to 3’ direction.
4.5 Polymerase Chain Reaction (PCR) in Practice
To synthesize DNA in the lab we need to perform PCR amplification of the DNA of interest using a plasmid vector or genomic DNA as template. For successful PCR amplification we have to cycle through three steps 25-30 times. The steps of PCR amplification are as follows:
Separation of the dsDNA template or strand separation
Annealing of the primers to the template DNA (they form Watson-Crick base pairs) to initiate DNA synthesis
DNA synthesis catalyzed by a polymerase
A graphic representation of a PCR cycle is seen on Fig. 4.5.