Unit 1 - Genetic Engineering Flashcards

Flashcards about genetic engineering techniques

Nuclease

Enzymes that cut, shorten, or degrade nucleic acid molecules by breaking the phosphodiester linkage.

Exonucleases

Nuclease that removes nucleotides from the ends of a DNA chain.

Bal31

An exonuclease of Alteromonas espejiana that removes nucleotides from both chains at the same time.

Exonuclease III

An exonuclease of E. coli that removes nucleotides from only one strand.

Endonucleases

Nuclease that breaks phosphodiester bonds internally.

S1 Nuclease

An endonuclease from Aspergillus oryzae that cuts short ssDNA.

DNase I

An endonuclease from Bovine pancreas that cuts dsDNA and ssDNA and is used in RNA extraction.

Restriction Endonucleases

A kind of endonuclease.

RNAse A

An endonuclease from Bovine pancreas that degrades ssRNA and is used in DNA extraction.

RNAse H

An endo- and exoribonuclease that is specific for RNA:DNA hybrids and is used in RT-PCR for RNA strand removal.

Polymerases

Enzymes that copy DNA molecules.

Thermostable Polymerases

Polymerases that catalyze the 5'-3' polymerization of DNA from a primer; examples include Taq and Pfu.

Taq Polymerase

A thermostable polymerase homologous to DNA Pol I of E. coli but lacks 3'-5' exonuclease domain (proofreading).

Pfu Polymerase

A thermostable polymerase that has a 3'-5'exonuclease domain (proofreading).

DNA Polymerase I (E. coli)

A polymerase that, in vivo, eliminates the RNA primer and fills in the gaps between Okazaki fragments.

Klenow Fragment

A polymerase that lacks 5'-3' exonuclease activity but maintains 3'-5' activity (proofreading).

Reverse Transcriptase (RT)

A polymerase with 5'-3' DNA polymerase activity on an RNA template.

RNA Polymerases

DNA-dependent RNA-dependent RNA polymerase activity, used in in vitro transcription.

Alkaline Phosphatase

An enzyme that removes the phosphate (P) group from a free 5’ DNA end; used in dephosphorylation prior to radiolabeling.

Polynucleotide Kinase

An enzyme that adds group P to free 5' end; used in radioactive labeling of probes and phosphorylation of oligonucleotides.

Terminal Transferase

An enzyme that adds terminal dNTP to the 3'OH end of dsDNA or ssDNA; used for homopolymer tailing.

Restriction Endonucleases

Enzymes that protect bacteria against bacteriophages & work with DNA methylases to degrade same sequence, protecting bacterial DNA

Type II Restriction Endonucleases

Domain is separated from endonuclease domain and are used in Genetic Engineering.

Isoschizomers

Enzymes that recognize the same target sequence, but do not necessarily break it at the same place.

Neoschizomers

Two enzymes (like Sma I or Xma I) which recognize the same sequence but cut in different places.

Star Activity

A Reduction of the specificity of sequence due to non optimal reaction conditions and/or if the reaction time is too long, the enzyme begins to cut in wrong sites.

DNA Ligases

Enzymes that repair breaks in one of the DNA strands (e.g. replication) and can also bind both DNA strands together.

T4 DNA Ligase

Derived from E. coli strains infected with T4 phage. Requires ATP and Mg2+ ions. The one which is most used due to a wider range of substrates: binds any DNA with blunt or cohesive ends; also binds RNA.

E. Coli DNA Ligase

Derived from E. coli strains infected with T4 phage. Requires NAD+ and Mg2+ ions. - Binds only dsDNA cohesive ends (no RNA).

Linkers

A short double-stranded DNA sequence with a recognition site for a specific restriction enzyme, used to add new restriction sites to a DNA fragment.

Adaptors

Short, chemically synthesized oligonucleotides with a pre-formed cohesive end that can be directly ligated to DNA fragments.

TOPO-TA Cloning

A ligation-free cloning technology utilizing Vaccinia virus DNA topoisomerase for direct insertion of Taq polymerase-amplified PCR products into a plasmid vector.

Recombinases

Enzymes that catalyze the reciprocal exchange of 2 dsDNA molecules, when at specific sequences are present in these molecules.

Gateway System

A commercial system based on the integrase (int) of phage l that facilitates efficient and high-throughput cloning through site-specific recombination.