Difference between revisions of "20.109(S21):M3D1"

Introduction

Though the theme of Module 1 is protein engineering, today will focus on a few key techniques used in DNA engineering. Because the sequence of proteins is determined by the sequence of the genes that encode them, learning how to manipulate DNA is an important first step. Today you will complete a cloning reaction to generate a protein expression vector that contains a gene that encodes a calcium-sensing protein. This process is illustrated in the schematic below. Later you will use this construct to engineer a new calcium-sensing protein.

Schematic of pRSET-IPC cloning. First, the EGFP insert is PCR amplified to generate multiple copies of the fragment that are flanked by restriction enzymes sites. Next, this fragment and the pRSET vector are digested to create compatible ends. Last, the compatible ends of the digested insert and vector are 'glued together' in a ligation reaction.

The cloning vector we will use is pRSET. This vector has several features that make it ideal for cloning and protein expression -- both of which are important for this module. The calcium-sensing protein we will study in Module 1 is inverse pericam (IPC). We will discuss this protein in much more detail later, for now it is sufficient to know that IPC was engineered to measure calcium concentrations. To generate your final product you will use three common DNA engineering techniques: PCR amplification, restriction enzyme digestion, and ligation.

Primer design

To amplify a specific sequence of DNA, you first need to design primers -- one primer that anneals at the start of the sequence of interest (the 5' end) and a second primer that anneals at the end of the sequence of interest (the 3' end). The primer that anneals at the start of the sequence is referred to as the 'forward' primer. The forward primer anneals to the non-coding DNA strand and reads toward, or into, the gene of interest. The 'reverse' primer anneals to the coding DNA strand at the end of the sequence and reads back into the sequence. Primers can also be useful in adding sequence to sequences upon amplification via the polymerase chain reaction.

Polymerase chain reaction (PCR)

The applications of PCR are widespread, from forensics to molecular biology to evolution, but the goal of any PCR is the same: to generate many copies of DNA from a single or a few specific sequence(s) (called the “template”). In addition to the template, PCR requires only three components: primers to bind sequence flanking the target, dNTPs to polymerize, and a heat-stable polymerase to catalyze the synthesis reaction over and over and over. DNA polymerases require short initiating pieces of DNA called primers to copy DNA. In PCR amplification, forward and reverse primers that target the non-coding and coding strands of DNA, respectively, are separated by a distance equal to the length of the DNA to be copied. To amplify DNA, the original DNA segment, or template DNA, is denatured using heat. This separates the strands and allows the primers to anneal to the template. Then polymerase extends from the primer to copy the template DNA. How many cycles of PCR are required to achieve the desired double-stranded amplification product?

Schematic of PCR amplification. PCR amplification results from multiple (typically ~30) cycles of three steps: denaturation, annealing, and extension.

Several features are important to consider when designing primers for PCR. Primers that are too short may lack requisite specificity for the desired sequence, and thus amplify an unrelated sequence. The longer a primer is, the more favorable are its energetics for annealing to the template DNA, due to increased hydrogen bonding. On the other hand, longer primers are more likely to form secondary structures such as hairpins, leading to inefficient template priming. Two other important features are G/C content and placement. Having a G or C base at the end of each primer increases priming efficiency, due to the greater energy of a GC pair compared to an AT pair. The latter decrease the stability of the primer-template complex. Overall G/C content should ideally be 50 +/- 10%, because long stretches of G/C or A/T bases are both difficult to copy. The G/C content also affects the melting temperature. PCR is a three-step process (denature, anneal, extend) and these steps are repeated 20 or more times. After 30 cycles of PCR, there could be as many as a billion copies of the original template sequence.

Restriction enzyme digest

Schematic of DNA digestion.

Restriction endonucleases, also called restriction enzymes, 'cut' or 'digest' DNA at specific sequences of bases. The restriction enzymes are named according to the prokaryotic organism from which they were isolated. For example, the restriction endonuclease EcoRI (pronounced “echo-are-one”) was originally isolated from E. coli giving it the “Eco” part of the name. “RI” indicates the particular version on the E. coli strain (RY13) and the fact that it was the first restriction enzyme isolated from this strain.

The sequence of DNA that is bound and cleaved by an endonuclease is called the recognition sequence or restriction site. These sequences are usually four or six base pairs long and palindromic, that is, they read the same 5’ to 3’ on the top and bottom strand of DNA. For example, the recognition sequence for EcoRI is 5’ GAATTC 3’ (see figure at right). EcoRI cleaves the phosphate backbone of DNA between the G and A of the recognition sequence, which generates overhangs or 'sticky ends' of double-stranded DNA.

Unlike EcoRI, some other restriction enzymes cut precisely in the middle of the palindromic DNA sequence, thus leaving no overhangs after digestion. The single-stranded overhangs resulting from DNA digestion by enzymes such as EcoRI are called sticky ends, while double-stranded ends resulting from digestion by enzymes such as HaeIII are called blunt ends. HaeIII recognizes 5’ GGCC 3’ and upon recognition cuts in the center of the sequence.

Ligation

Schematic of DNA ligation.

In a ligation reaction, DNA ends are covalently attached to one another via the ligase enzyme. The efficiency of the reaction is related to type of DNA ends: compatible sticky ends will ligate more efficiently than blunt ends, and non-compatible sticky ends will not be ligated due to the lack of hydrogen bonding between the basepairs. To initiate the ligation reaction, hydrogen bonds are formed between the compatible overhangs of DNA fragments. The ligase enzyme then forms a covalent phosphodiester bond between the 3' hydroxyl end of the 'acceptor' nucleotide and the 5' phosphodiester end of the 'donor' nucleotide.

The first step in this process is the addition of AMP (adenylation) to a lysine residue within the active site of DNA ligase, which releases a pyrophosphate. Next, the AMP is transferred to the 5' phosphate of the donor nucleotide resulting in the formation of a pyrophosphate bond. Lastly, a phosphodiester bond is formed between the 5' phosphate of the donor nucleotide and the 3' hydroxyl of the 3' acceptor nucleotide.

Protocols

Reagents list

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Next day: Examine IPC mutations