Difference between revisions of "20.109(S16):In situ cloning (Day1)"

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. The EGFP insert is PCR amplified then ligated into the pRSET vector using a compatible restriction enzyme recognition sequence, EcoRI.

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 this module is inverse pericam (IPC). We will discuss this protein in much more detail later, for now it is sufficient to know that is used to measure calcium concentrations. To generate your final product you will use three common DNA engineering techniques: PCR amplification, restriction enzyme digestion, and ligation.

PCR amplification

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 “target” or “template”).

In addition to the target, PCR requires only three components: primers to bind sequence flanking the target, dNTPs to polymerize, and a heat-stable polymerase to carry out the synthesis reaction over and over and over. 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 target sequence.

Kary Mullis.

Based on the numerous applications of PCR, it may seem that the technique has been around forever. In fact it is just over 30 years old. In 1984, Kary Mullis described this technique for amplifying DNA of known or unknown sequence, realizing immediately the significance of his insight.

"Dear Thor!," I exclaimed. I had solved the most annoying problems in DNA chemistry in a single lightening bolt. Abundance and distinction. With two oligonucleotides, DNA polymerase, and the four nucleosidetriphosphates I could make as much of a DNA sequence as I wanted and I could make it on a fragment of a specific size that I could distinguish easily. Somehow, I thought, it had to be an illusion. Otherwise it would change DNA chemistry forever. Otherwise it would make me famous. It was too easy. Someone else would have done it and I would surely have heard of it. We would be doing it all the time. What was I failing to see? "Jennifer, wake up. I've thought of something incredible." --Kary Mullis from his Nobel lecture; December 8, 1983

Restriction enzyme digest

Restriction enzyme digest with EcoRI. EcoRI cuts between the G and the A on each strand of DNA, leaving a single stranded DNA overhang (also called a “sticky end”) when the strands separate.

Restriction endonucleases, also called restriction enzymes, cut (“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 (see also figure at right) is

5’ GAATTC 3’
3’ CTTAAG 5’

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’
3’ CCGG 5’

Ligation

During the ligation reactions, hydrogen bonds will form between the overhangs on the fragments, and then the ligase will repair the phosphate backbone, creating a stable circular plasmid (as shown in the figure below).

Schematic of DNA ligation.

Protocols

Because DNA engineering at the benchtop can take days, if not weeks, you will generate your clone in silico today. Please document your work and answer all questions in your notebook as you progress through the exercises below.

Part 1: PCR amplification and restriction enzyme digest of IPC insert

PCR amplification and restriction enzyme digest of IPC insert.

Part 2: Restriction enzyme digest of pRSET vector

Restriction enzyme digest of pRSET vector.

Part 3: Ligation of IPC insert and pRSET vector

Ligation of IPC insert and pRSET vector.

Part 4: Confirmation digest

Navigation links

Next day: Design mutation primers