lecture 2 Flashcards
where is most DNA found?
right helix
what is polymerase chain reaction (PCR)?
-amplification (exact copying) of DNA
-adds bases onto 3’ end
what does PCR produce?
new strand of DNA complimentary to the original template strand
how does replication occur in prokaryotes?
-enzyme helicase (by using energy) separates the ds DNA to form replication fork
-breaks the Watson crick bonds
-primases attach RNA to open strands to form natural primer
-DNA polymerase attaches to the double strand (at one end)
-slides along 5’ to 3’ of leading strand to produce complimentary strand
-produces one long strand of DNA
-product also produced for complimentary to lagging strand
-primase comes along to add another primer and form small DNA fragments
-ligase comes and ligates strands together forming phosphate backbone
-continuous piece of DNA formed
what is needed for replication in eukaryotic cells?
-template DNA
-40 or more proteins
-helicase
-primase (initiation)
-polymerases (Alpha, delta and epsilon)
-nuclease
-ligase
-ssDNA binding proteins
-sliding clamps
what does the alpha polymerase do?
initiates the process of leading strand
what does the epsilon polymerase do?
replicates the lagging strand
what does the delta polymerase do?
takes over from alpha polymerase, produces the end product
why do we use PCR?
sensitive, specific, cheap, rapid, robust (DNA is very stable)
why does PCR require no added energy?
dNTP is an energy storage molecule so provides the energy for the reaction
what occurs in the DNA synthesis reaction?
-hydrolysis of phosphorus dimer at end
-phosphate released and binds to hydroxyl group on previous ribose sugar to form phosphodiester bond
what is in the reaction?
template, 2 primers (to prime synthesis), polymerase (copies template of DNA), dNTPs, magnesium (cofactor for DNA polymerase), buffer (maintains pH)
what do the nucleotides add to the structure?
strength (phosphate) and specificity (from base) and helps with orientation
what is the function of taq polymerase?
-accurately copies DNA
why use taq polymerase?
has a high conc at high temperatures- doesn’t denature within the process and temp changes
what are the region of the enzyme?
synthesis, proof reading and primer removal
what occurs in the synthesis region?
-where protein binds two DNA and synthesises by forming phosphodiester bonds
what occurs in the proof reading region?
-makes sure the right nucleotides come and bind at the correct time
-checks if Watson crick bonds are formed accurately
what are the characteristics needed for a primer?
-single stranded DNA (oligonucleotide)
-18-24 bases in length (still specific but hybridises quickly)
-high GC content to provide strong attachment and bonds (start and end with GC)
-annealing point/ melting temp of 50-60 degrees
-primer pairs have difference of 5 degrees so can happen in same cycle (efficient)
-primer pairs cant have complimentary region as they’ll bind together
why is magnesium used?
-cofactor to maintain activity of primer
-enables activity of the catalysis
-enhances enzymatic activity and supports dna application
where does the magnesium bind to?
-active site and enables the binding of a protein
what is the purpose of the buffer?
-promotes the annealing of primers to the DNA
what are the three stages of the PCR reaction?
-denaturation
-annealing
-elongation
What happens at 92 C in PCR?
DNA hydrogen bonds between double strands break apart
