L3 - Amplifying DNA: PCR Flashcards
How was DNA amplified before PCR?
In bacterial cells
E. coli cells, a vessel/machine to amplify DNA
Typically a plasmid – small circular DNA molecule conferring antibiotic resistance
Works well & is still used today
takes 48 hours to amplify
What is PCR?
A single enzyme, rather than a cell to amplify DNA
Enzyme = DNA polymerase
Other biochemicals are necessary
Works well – quick (2 hours) & powerful
DNA replication in cells
Semi conservative – both strands are a template
5’ to 3’
DNA replication in eukaryotic cells
What is needed?
Template DNA Primase Polymerases Helicases Nucleases Ligases ssDAN binding proteins Sliding clamps
What does primase do in eukaryotic DNA replication?
Initiates synthesis
What are the types of polymerases involved in eukaryotic DNA replication?
DNA polymerase is an enzyme that synthesizes DNA molecules from deoxyribonucleotides, the building blocks of DNA
Alpha – makes a lot of mistakes and works on RNA as well
Delta
Epsilon – has a PCNA ring
What do helicases do in eukaryotic DNA replication?
Unwinding DNA to provide the substrates for polymerases
What do ligases do in eukaryotic DNA replication?
Ligate DNA together
What do ssDNA binding proteins do in eukaryotic DNA replication?
DNA is fragile by itself
Duplication of DNA in a test tube
Ingredients
Template – ssDNA
Primer, to prime synthesis – small ssDNA molecule, 6-30 bases, chemically synthesised – anneals to DNA and mimics primase
Polymerase – to copy the template, extending from the 3’ end of primer
dNTPs – deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, dTTG)
Magnesium – critical co-factor for DNA polymerase enzyme
Buffer to maintain pH & provide necessary salt – mimics intracellular conditions
Duplication of DNA in a test tube
Process
Add all the ingredients at correct concentrations
Incubate & leave for polymerase enzyme to work
Product = replicated dsDNA
Why do you boil the DNA when making DNA in a test tube?
Disrupts the base pair bonding
How do you get the duplication of both strands of DNA in a test tube?
2 primers in different directions
Other primer will make the complementary strand
In a single reaction you end up with 2 ds strands
Timings of PCR
30s 95 degrees
60s 55 degrees
60s 72 degrees
Polymerases used are from heat tolerant bacteria – don’t get denatured by the boiling
Detection of PCR products
Run products on agarose gel
Use intercalating dye to stain DNA to determine size & yield
