BIOL1951 #5 - Genetic Technology (1)

Recombinant DNA

Artificial DNA strand created by joining combining two or more gene sequences

Polymerase Chain Reaction (PCR)

In vitro form of DNA Replication using heat-stable Taq Polymerase

PCR in vitro vs. DNA Replication in cell

1. Denaturization w/ temperature ➡ Helicase

2. DNA Primer ➡ RNA Primer

3. Taq Polymerase ➡ DNA Polymerase

Oligonucleotide Primer

Short, single-stranded DNA sequences that bind to template DNA to start replication

Gel Electrophoresis

• Technique used to separate mixtures of nucleic acids/proteins based on their size and electrical charge

  • Smaller molecules travel faster and farther down the gel

Vector

• A DNA molecule into which “foreign” DNA is inserted

  • Ex. plasmids, cosmids, etc.

Types of Vectors

1. Cloning Vectors

2. Expression Vectors

3. Integration Vectors

Key Features of Expression Plasmids

• Origin of Replication (ori)

• Selectable Marker (amp)

• Multiple Cloning Site (MCS)

Origin of Replication (ori)

Binding site of DNA Polymerase

Selectable Marker (amp)

Ensures the plasmid is successfully implemented ➡ Resistance to amplicillin

Multiple Cloning Site (MCS)

Provides a precise insertion site for the foreign gene of interest

Restriction Enzyme

• Proteins that cut DNA at specific sequences (restriction site)

  • Almost always palindromic

Types of Restriction Enzymes

1. Some restriction enzymes make staggered cuts ➡ Produce single-stranded ends

2. Some restriction enzymes make non-staggered cuts ➡ Produce double-stranded  "blunt" ends

• More difficult to form bonds with

Restriction Site

Specific DNA sequence, usually 4-8 nucleotides long, recognized and cleaved by restriction enzymes

Palindome

Nucleic acid sequence that reading from 5’ to 3’ on one strand matches the 5’ to 3’ sequence on the complementary strand

Plasmid Vector Process

1. Vector is cut by the Restriction Enzyme at the MCS

2. Gene of interest hydrogen-bonds to the sticky-ends of the MCS

3. DNA Ligase catalyzes covalent bonds