semi-conservative replication meaning
one strand of parent DNA is kept in daughter molecule called the “template strand”
other half is determined by the code on the template strand and is built from free nucleotide
importance of DNA replication
growth
replacement
reproduction
importance of original strand
ensures genetic continuity with high degree of accuracy so cells produced inherit correct genetic sequence
Crick and Watson
made hypothesis about how DNA is copied during growth
proposed semi-conservative model but with no evidence
evidence later provided by Meselesonn and Stahl
process of semi-conservative replication
helicase unwinds DNA (flattening helix structure)
helicase causes bonds to break, expossing bases
each polynucleotide strand acts as a template for the formation of a new strand made from free nucleotides
DNA links nucleotides together to form new strand
that is done by DNA Polymerase which catalyses the condensation reactions linking deoxyribose sugar and phosphate group
always built in 5 to 3 direction
then hydrogen bonds form between complementary base pairs
gel electrtophoresis
technique used widely in analysis of DNA, RNA, and proteins
molecules separate with an electric current according to size or mass and net overall charfe
why does seperation occur
electrical charge - positive move to cathode, negative to anode
size of molecules - move at different rates
type of gel
DNA separation preparation
DNA must be prepared for gel electrophoresis so it can be sequenced or analysed for genetic profiling
to prepare, scientists must amplify number of DNA molecules by polymerase chain reaction
then the restriction enzyme chop DNA into fragments
method of gel electrophoresis
create agarose gel plate and add wells in one end
submerge gel in an electrolyte solution
load DNA fragments with micropipette
apply electric current
not visible yet - transfer to absorbent platter and head to separate 2 DNA strands
probes added to develop visual output: radioactive label, fluorescent stain or dye
polymerase chain reaction (PCR)
in vitro method of DNA amplification
used to produce large quantities of specific fragments of DNA or RNA from small quantity
requirements of PCR
target RNA/DNA sequence (whole genome not required) - section identified by primer sequence
DNA polymerase Taq Polymerase because it doesn’t denature at high temperatures
Free nucleotides
Buffer solution for optimum PH
key stages of PCR
3 stages per cycle
each cycle DNA is doubled
PCR occurs in thermal cycler which produces optimum temperatures and controls length of each stage
denaturation . heated to 95, breaks hydrogen bonds
annealing - decreased to 50-60 so primer anneals to ends of single strands
elongation - increased to 72 for optimum temperature for Taq polymerase to build new strand
Applications of electropherosis
every person has a repeating, short, non coding region of DNA unique to them
VNTR (variable number tandem repeats)
can be used in forensic investigation, identifying corpses, or paternity tests
directionality of DNA polymerase
replication must occur in 5 to 3 direction
DNA polymerase works in 5 to 3 direction adding nucleotides to 3 end
leading vs lagging strands
leading: continuously following fork
lagging: discontinuously in Okazaki fragments away from the fork
Okazaki fragmenrs are later joined together by DNA ligase
before DNA can be added to new strand RNA primer must be added to create a binding point
added once to leading strand and in fragments to lagging strand
enzymes in DNA replication
helicase: unwinds and causes hydrogen bonds to break
single stranded binding proteins: keep strands seperated
dna primase III: acts as a proofreader and removes incorrect nucleotides, starts replication next to RNA primer
dna primase I: removes RNA primer and replaces with DNA
dna ligase: joins Okazaki fragments