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Polymerase Chain Reaction

Exploits the activity of DNA polymerases to amplify specific regions of DNA


identified by primers, small segments of DNA that act as the origin of amplification.
Made possible by the discovery of thermostable polymerases from thermophiles.
Dependent upon the melting temperatures and annealing temperatures of DNA.
High temperatures (~98C) are used to denature (separate) DNA strands
completely.
Primers annealed to template strands at 50-70C, depending upon the
composition and length of the primers. The Tm will increase as the GC content and
length increase.
The polymerase elongates the complementary strand off of the primer in a
5 to 3 direction as it reads the template strand in a 3 to 5 direction when at 72C.
This temperature cycling, which was originally achieved through the use of
multiple hot baths, is now automated through scientific instruments called
Thermocyclers
Molecular Cloning
Method by which cDNA or a gene is obtained: the polyA tail of mRNA can be used to
capture all produced, intron-free mRNA, which are then transcribed into ss DNA by
reverse transcriptases.
Site Directed Mutagenesis
SDM refers to the alteration of a single nucleotide, an entire amino acid (codon), or
several amino acids.
Both methods utilize primers with complete overlap with the template strand with high
enough melting temperatures in order to compensate for the disruption at the site of the
base pair mismatch/mutants.
The main difference between the Stratagene and NEB protocols is linear vs exponential
amplification of the new DNA, and the primer changes that enable that.
Quikchange
Primers: Completely overlapping, mutation in center.
Phosphorylation: Not required

Amplification: Linear
DPN1 Digest: Required
Ligation: Not Required
NEB Exponential
Primers: Back-to-back, mutation at center of one
Phosphorylation: Required
Amplification: Exponential
DPN1 Digest: Not Required
Ligation: Required
Sub-cloning
Sub cloning entails the transfer of a gene from cDNA to an expression vector, or the
transfer of genes between expression vectors.
Multiple types of sub cloning are utilized, but the most common type makes use of
restriction enzymes for the highly-specific splicing of DNA fragments
Restriction Enzymes
Utilizes common Restriction sites (i.e. NdeI, NcoI, HindIII, BamHI, NotI,
XhoI, EcoRI) and corresponding enzymes to cut and paste DNA fragments.
Overlap extension PCR can be used to amplify DNA while also adding on
restriction sites. Additional bases should be added before site to maximize efficiency, as
shown here: https://www.neb.com/tools-and-resources/usage-guidelines/cleavageclose-to-the-end-of-dna-fragments.
Advantage: Widely used, diverse options. Disadvantage: Complicated,
may use low efficiency enzymes
Gateway
Gene is transferred from a vector or cDNA into a donor Gateway vectors
using restriction enzymes which can easily transfer the gene to recipient Gateway
vectors.
Advantage: Simplicity. Disadvantage: Lack of diversity in options

TOPO/TA
Gene is amplified using Taq-driven PCR, which leaves an adenosine
nucleotide at the 3 end. TOPO vectors are sold ready-to-ligate with a 5 T overhang and
a 3-fused DNA topoisomerase
Advantage: Very quick and easy. Disadvantage: Cost, Lack of diversity,
Vectors can only be ordered
Isothermal Assembly Reaction (Gibson Assembly)
Uses a 5 exonuclease to digest 5 ends of any dsDNA. Homologous
regions will auto anneal. A polymerase will fill in ssDNA regions and a ligase will fix any
nicks.
Advantage: Very diverse in application as it does not require specific
sequences. Disadvantage:
Type IIS Assembly (Golden Gate/MoClo)
Ligation independent cloning (LIC)
Yeast Mediated/Oligonucleotide Stitching

Vector Development
Origins of Replication
Antibiotic Resistance
MCS cassettes
Phosphorylation
DNA Polymerases
Restriction

Dephosphorylation
Ligation
Purification
Gel
Silica
Transformation
Cell Choice