DNA Cloning: Restriction Enzymes

• DNA cloning involves molecular techniques, genetic engineering, and recombinant DNA technology
• It is a molecular technique that makes many identical copies of a DNA piece (gene) by introducing it into a replicating host cell via a vector
• Essential properties of a vector includes capacity for autonomous replication within a host cell and the presence of a specific nucleotide sequence recognized by restriction endonucleases
• Plasmids are the most commonly used vectors

Restriction Enzymes

• Restriction enzymes are DNA-cutting enzymes that recognize a specific target sequence, known as the recognition site
• They cut the DNA into two pieces at or near that recognition site, creating a restriction site
• The cut DNA pieces can be reformed again without ligase enzyme
• Discovered from bacteria as a defense mechanism to limit expression of nonbacterial DNA
• They degrade cut DNA using exonucleases and prevent phage infection
• Bacterial DNA is methylated at A or C at recognition site to protect it from being recognized and cleaved by its own restriction enzymes
• Named according to the species from which they are derived
• EcoRI comes from Escherichia coli (E. Coli), and the 'R' represents the specific strain from which it was isolated
• Restriction enzymes are also listed by order discovered, e.g. EcoRI, EcoRII

Types of Restriction Enzymes

• Type I restriction enzymes cleave randomly up to 1000 bp away from the recognition site
• Type II restriction enzymes cleave within or near the recognition site with no ATP needed
• Type III restriction enzymes cleave randomly 24-26 bp downstream of the recognition site

Restriction Enzyme Specificity

• Majority Type IIP enzymes recognize and cleave within the same palindromic recognition sequence
• Minority Type IIS enzymes cleave at asymmetric target sites some distance away from their recognition sites
• Isochizomers are restriction enzymes that have the same recognition sequence and cleavage site
• Neochizomers are restriction enzymes that have the same recognition sequence but cleave at a different site

Double Digest

• Double digest involves using two restriction enzymes together to increase efficiency
• One can mix enzymes in the same compatible buffers
• Sequential digests can be conducted
• Some enzymes display "star activity" in certain buffers, causing them to digest the DNA at sites other than the standard recognition site
• High-fidelity (HF) restriction enzymes have 100% activity in CutSmart buffer with reduced star activity

Plasmids as Vectors

• Plasmids are small, extrachromosomal, circular, and supercoiled DNA molecules that can replicate independently
• Bacteria gain new traits like antibiotic resistance using the cloning vector in the plasmid
• The plasmid consists of the origin of replication, antibiotic resistance gene, selectible marker, and multiple cloning site

Plasmid Structure: Origin of Replication

• Origin of replication (replicon) is a critical site used by bacteria to start plasmid replication.
• In mammalian cells, due to the complexity of replication, the replication for plasmids is limited by the number of plasmids delivered to the cell
• Transient transfection dilutes the plasmid copy number via cell replication
• Plasmids are classified into relaxed (replication depends on DNA on plasmid itself only, high copy number) and stringent (regulated by host DNA, low copy number)

Plasmid Vectors

• Plasmids can have > 1 origin of replication (called a shuttle vector) to allow propagation in different host organisms
• SV40 ORI allows autonomous replication (as an episome) in mammalian cells expressing the SV40 large T-antigen

Selectable Markers On Plasmids

• Selectable markers allows for selection of transformed cells that uptook the plasmid and became resistant to the marker (usually antibiotic resistance gene)
• Antibiotic resistance gene present in bacteria (e.g., Ampicillin, gene b-lactamase)
• Eukaryotic selectable markers include Puromycin, Blasticidin, Hygromycin B, bleomycin

Multiple Cloning Site (MCS)

• The multiple cloning site (MCS), also known as a polylinker, is a synthetic DNA fragment containing many recognition and restriction sites for various restriction enzymes
• Synthetic cloned DNA fragment containing recognition restriction sites for many restriction enzymes

Eukaryotic Promoters

• Eukaryotic promoter is approximately 100 to 1000 bp upstream of the gene
• Transcription is carried out by: RNA Pol I (Ribosomes), RNA Pol II (mRNA for protein production), and RNA Pol III (small non-coding RNA)
• The promoter must be compatible with the polymerase needed to bind
• General expression promoters (mRNA): CMV, SV40, CAG, human beta actin, Ubc, EF1a
• Small RNA expression promoters include U6 and H1

Types of Promoters

• Constitutive promoters are expressed at a constant rate (e.g., H1)
• Inducible promoters are expressed/induced by a certain condition, e.g., H1Tet ON where the gene is only induced when cells are treated with tetracycline (e.g., Doxycycline)

Internal Ribosome Entry Site (IRES)

• Multicistronic vectors use an Internal Ribosome Entry Site (IRES) to produce multiple proteins from a single mRNA transcript
• 2A peptides (LINKER) are now also used to produce multicistrone proteins

Translation Initiation and Termination

• Eukaryotic Translation Initiation: Kozak consensus sequence (5'-GCCRCCAUGG-3')
• Prokaryotic Translation Initiation: Shine–Dalgarno (SD) sequence (5'-AGGAGG-3')
• Termination signal is complicated in Eukaryotes for pol III, requiring a poly-dT sequence (6-9 bases long) in the sense strand