Genetic Engineering: DNA Polymerases and Replication

Molecular Genetics 2022

• This is a course on Molecular Genetics offered at MISR University for Science & Technology.

Vision of the Biotechnology Department

• The department aims to be an academically accredited and pioneering body in the field of biotechnology, both regionally and internationally.

Mission of the Biotechnology Department

• The department is committed to producing qualified biotechnology engineers according to established academic standards.
• It intends to meet the needs of the local and regional labor market in the medical, pharmaceutical, agricultural, and environmental sectors.
• It is also focused on conducting innovative scientific research, providing community service, and offering scientific consultations within an upwardly mobile mindset.

DNA Manipulative Enzymes

• Intended Learning Outcomes (ILOs):
  • Describe the activity and main applications of different enzymes used in recombinant DNA research.
  • Differentiate between different types of DNA manipulative enzymes.

DNA Manipulation

• The key activities of DNA manipulation include cutting and joining DNA, shortening or lengthening DNA, altering it by adding or removing chemical groups, and copying it into RNA or DNA.

DNA Manipulative Enzymes

• DNA manipulative enzymes are grouped into four broad classes based on their catalyzed reactions:
  • Nucleases: cut, shorten, or degrade nucleic acid molecules.
  • Ligases: join nucleic acid molecules together.
  • Polymerases: make copies of molecules.
  • Modifying enzymes: remove or add chemical groups.

Nucleases

• Nucleases break phosphodiester bonds linking nucleotides in a DNA strand.

• Two main types of nucleases are:

  • Exonucleases: remove nucleotides one by one from the end of a DNA molecule.
  • Endonucleases: break internal phosphodiester bonds within a DNA molecule.

• Differences in exonucleases: the distinguishing factor is the number of strands degraded in a double-stranded molecule.

• Example exonuclease: Bal31 removes nucleotides from both strands of a double-stranded molecule.

• Other example exonuclease: Exonuclease III degrades one strand of a double-stranded molecule, leaving the other strand intact.

• Example endonucleases:

  • S1 nuclease only cleaves single strands.
  • DNase 1 is non-specific, attacking DNA at any internal phosphodiester bond, resulting in a mixture of mononucleotides and short oligonucleotides after prolonged action.
  • Restriction endonucleases cleave double-stranded DNA at specific recognition sites.

Ligases

• DNA ligase's cellular role is the repair of single-stranded breaks in double-stranded DNA molecules, like during replication.
• They also link individual fragments of double-stranded DNA together.

Polymerases

• DNA polymerases synthesize new DNA strands complementary to an existing DNA or RNA template.

• Four types of DNA polymerases are used in genetic engineering.

• DNA polymerase I: a dual activity enzyme conducting DNA polymerization and degradation processes.

  • The Klenow fragment is a modification of DNA polymerase I, retaining polymerase function but lacking nuclease activity. Used for DNA synthesis on single-stranded templates.

• Taq DNA polymerase: Used in the polymerase chain reaction (PCR). It's an enzyme from the bacterium Thermus aquaticus.

• Reverse transcriptase: produces cDNA (complementary DNA) from RNA templates.

DNA Modifying Enzymes

• These enzymes are:
  • Alkaline phosphatase: a hydrolase that removes phosphate groups from molecules. Used to prevent DNA ligation and in radiolabeling of DNA
  • Various types of alkaline phosphatases are common in research:
    • Bacterial alkaline phosphatase (BAP)
    • Shrimp alkaline phosphatase (SAP)
    • Calf intestine alkaline phosphatase (CIAP)
    • Placental alkaline phosphatase (PLAP)
  • Polynucleotide kinase: adds phosphate groups onto free 5’ termini, the reverse function of alkaline phosphatase
  • Terminal deoxynucleotidyl transferase: catalyzes adding one or more deoxyribonucleotides to the 3' terminus of a DNA molecule, independent of a template. This enzyme has use in rapid amplification of cDNA ends (RACE), and adding radioactively labeled nucleotides.

Topoisomerases

• Topoisomerases change the conformation of circular DNA, such as plasmids, by introducing or removing supercoils. This involves wrapping around DNA, making a cut, permitting the helix to spin, and reconnecting the broken strands.