Unit 3. - Genetic Engineering Techniques Flashcards

Flashcards covering key vocabulary and concepts related to genetic engineering techniques, with a focus on enzymes used for DNA manipulation in vitro.

Nucleases

Enzymes that cut, shorten, or degrade nucleic acid molecules by breaking the phosphodiester linkage between the phosphate group at the C-5' position and the OH group at the C-3' position.

Exonucleases

Nucleases that remove nucleotides from the ends of a DNA chain.

Endonucleases

Nucleases that break phosphodiester bonds internally within a nucleic acid molecule.

Bal31 Nuclease

An exonuclease that degrades both 3' and 5' termini of duplex DNA and cleaves at nicks, gaps, and single-stranded regions.

Exonuclease III

An exonuclease that removes nucleotides from only one strand of DNA.

Lambda Exonuclease

A highly processive 5' to 3' exonuclease that removes 5' mononucleotides from duplex DNA, preferring 5'-phosphorylated double-stranded DNA.

S1 Nuclease

An endonuclease that shortens single-stranded DNA.

DNase I

An endonuclease that cuts double-stranded and single-stranded DNA; used in RNA extraction.

Restriction Endonucleases

Endonucleases that recognize and cleave DNA at specific sequences; used extensively in genetic engineering.

RNAse A

A nuclease that degrades single-stranded RNA; used in DNA extraction.

RNAse H

An endo- and exoribonuclease with activity in both the 3' to 5' and 5' to 3' directions, specific for RNA:DNA hybrids; used in RT-PCR for RNA strand removal.

Polymerases

Enzymes that synthesize new DNA or RNA strands on a DNA or RNA template

Thermostable Polymerases

Polymerases that catalyze the 5'-3' polymerization of DNA from a primer and are stable at high temperatures (e.g., Taq DNA polymerase, Pfu DNA polymerase).

Taq DNA Polymerase

A thermostable DNA polymerase homologous to DNA Pol I of E. coli but lacking a 3'-5' exonuclease domain.

Pfu DNA Polymerase

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

DNA Polymerase I (E. coli)

A polymerase that, in vivo, eliminates RNA primers and fills gaps between Okazaki fragments; possesses 5'-3' polymerase activity, 3'-5' exonuclease activity, and 5'-3' exonuclease activity.

Klenow Fragment

A fragment of DNA Polymerase I that retains polymerase activity and 3'-5' exonuclease activity (proofreading) but lacks 5'-3' exonuclease activity.

Reverse Transcriptase (RT)

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

Alkaline Phosphatase

An enzyme that removes the phosphate (P) group from a free 5' DNA end; used prior to radiolabeling and to prevent vector religation during cloning.

Polynucleotide Kinase

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

Terminal Transferase

An enzyme that adds terminal dNTPs to the 3'OH end of dsDNA or ssDNA; used in homopolymer tailing and radioactive labeling.

DNA Ligase

An enzyme that seals breaks in DNA strands by catalyzing the formation of a phosphodiester bond.

T4 DNA Ligase

A DNA ligase derived from E. coli strains infected with T4 phage that requires ATP and Mg2+ ions; binds any DNA with blunt or cohesive ends and also binds RNA.

E. coli DNA Ligase

A DNA ligase that requires NAD+ and Mg2+ ions; binds only dsDNA cohesive ends (no RNA).

Topoisomerases

Enzymes that cut and rejoin DNA strands to manage DNA topology; used in topoisomerase ligation for simultaneous DNA cutting and joining.

Recombinases

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

Linkers

Short, double-stranded DNA fragments with a specific restriction enzyme site, used to add restriction sites to blunt-ended DNA fragments.

Adaptors

Short, double-stranded DNA fragments with a pre-made cohesive end, ligated to blunt-ended DNA fragments.