Enzymes used in this lab

We will be using several enzymes in the 305 lab. It is important that you be aware of each enzymes source and optimal conditions. Except where noted, all enzymes are purchased from Thermoscientific;
please go to the Thermoscientific site and read each enzyme's information sheet.

Restriction enzymes

Type II restriction enzymes, the type generally used in cloning, recognize specific DNA sequences and cut the DNA in exact positions relative to the recognition sequence. The cut site can either be symmetrical (leaves blunt ends – no single strand DNA overhangs) or asymmetrical (leaves DNA overhangs commonly called sticky ends). In the case of sticky ends, the two pieces of DNA must be complementary in order for the fragments to be joined together by the enzyme DNA ligase (more on ligation later). This is why, ideally, we want the restriction enzymes that we use for excising the DNA we are cloning to also cut the vector within the MCS. Refer to Figure 1 for a representative diagram of restriction enzyme recognition sites and the products of digestion.

rest_enz.jpeg - click on image for larger view.
Figure 1. Cartoon showing the restriction products produced when DNA is digested with EcoRI (top panel) and EcoRV (lower panel) enzymes. The DNA sequence recognized by the enzyme is indicated in block letters; the dotted line to either site of the recognition sequence indicates an unspecified number of nucleotides may be present in the DNA molecule on either side of the recognition site. 5' indicates the position of a 5' phosphate at the end of the DNA molecules; the opposite strand has a 3' OH group at the same end (not shown). The arrows pointing at the recognition site indicate where the enzyme will cleave the DNA molecule. DNA molecules produced by each restriction enzyme are indicated to the left of the large grey arrow. The position of the 3'-OH and the 5'-P left at the ends of the new DNA molecules are indicated. Digestion with EcoRI produces DNA molecules with single stranded overhangs: these are called sticky-ends. Digestion with EcoRV does not produce single-strand overhang: these are called blunt-ends.

Taq polymerase

Taq polymerase [2] is a thermostable DNA polymerase used to synthesize DNA copies in PCR reactions. It gets its name from the bacteria it was originally isolated from, Thermus aquaticus [1]). PCR reactions must have thermostable polymerases that can tolerate the repeated high temperature cycling (up to 96 °C).

DNA Ligase

In vitro, DNA ligase catalyzes the formation of phosphodiester bonds between the 5' phosphate and the 3' hydroxyl termini in duplex DNA or RNA. It is an essential enzyme for DNA replication because it joins the Okazaki fragments that are produced during lagging strand DNA synthesis.

In molecular cloning, DNA ligase is used to join either blunt or cohesive DNA ends together. The DNA ends must be double stranded and close together, and there must be ATP in the reaction. We will be using T4 DNA ligase.

Citations
2. Chien A, Edgar DB, Trela JM. 1976. Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. J Bacteriol. 127(3):1550-7.
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