Lecture 2 - Primers, Vectors & Restriction Enzymes Flashcards
What are primers?
Short pieces of single-stranded DNA that are complementary to the target sequence (on template strand)
Provide a starting point for DNA synthesis
What are primers used for?
Cloning
Quantification of gene expression
Mutagenesis - place mutated sequence near the middle of the primer, or at least 7-8 nucleutides away from the 3’ end
Sequencing
What are the 5 considerations in PCR primer design?
Primer length
Primer melting temperature (Tm)
Primer annealing temperature (Ta)
Primer GC content and CG Clamp
Insert restriction enzyme cut sites
How should primer length be decided?
Optimally 18-24 bp - short enough to bind easily, long enough for adequate specificity. Affects Tm and Ta
What is the primer melting temperature (Tm) of a primer?
Temperature at which half of primers are bound, optimally 50-60°C (within 5°C for each primer pair). Every G or C = 4°C. Every A or T = 2°C.
What is the primer annealing temperature (Ta) of a primer?
Optimal annealing temperature experimentally determined. 5-10°C lower than melting temperature. Too high, primers might not bind effectively. Too low, primers may bind non-specifically
What should primer GC content be and why is a CG Clamp needed?
GC content between 40-60%. In the last 5 bases at 3’ end, there should at least be 2 G or C bases (GC Clamp) to increase stability of primer and improve specificity of primer binding
How to insert restriction enzyme cut sites?
Near 5’ end of primer. If restriction enzyme cut sites in primer, add 3-5 bases to the 5’ cut site. This is known as the leader sequence and allows for more efficient enzyme cutting
What are the don’ts in primer design?
Repeats/runs - four or more of a single base/dinucleotide repeats cause mispriming
Sequences that are like target (cross homology) - primer may bind to wrong region leading to mispriming. Check for cross homology using NCBI BLAST software
Primer scondary structure - fold in on themselves or bind to each other forming primer dimers, reduce availability of “good” primers
What is the use of the vector?
It is a vehicle to protect the DNA fragment, deliver it and make it functional in the host cell
Cloning vector acts as a vehicle to transfer the desired piece of DNA into the target cells
What are some applications of cloning vectors?
- Transfer and expression of transgene into recombinant protein in host cell
- Confer special characteristics to host (e.g., metabolic, antibiotic resistance)
- Controlling expression of cellular genes
- Preservation of DNA fragments in genome library construction
A cloning vector must have 4 following characteristics:
Ability to replicate in a specific host cell (e.g., bacteria, yeast, plant, mammalian cell, etc.)
Contains a genetic marker for selection of cells carrying plasmid.
Unique restriction enzyme sites to allow insertion of foreign DNA.
Minimum amount of nonessential DNA sequences
What are plasmids?
- Plasmids are extrachromosomal circular, self-replicating, double-stranded DNA molecules present in bacteria
- Plasmid along with genomic DNA are passed on to the next generation of daughter cells
How do plasmids replicate?
Rolling circle mechanism
What is the function of Ori sites?
- Ori sites allow for efficient recruitment of the host replication machinery
- Specific to bacteria species or non-specific to a broad range in multiple bacterial species
- Often more than one Ori is included in plasmids to permit their use in more than one host cell
- A plasmid vector can be high or low copy number based on its replication efficiency
