Biology - DNA replication

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

1. helicase unwinds DNA (flattening helix structure)

2. helicase causes bonds to break, expossing bases

3. each polynucleotide strand acts as a template for the formation of a new strand made from free nucleotides

4. DNA links nucleotides together to form new strand

5. that is done by DNA Polymerase which catalyses the condensation reactions linking deoxyribose sugar and phosphate group

6. always built in 5 to 3 direction

7. 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

1. electrical charge - positive move to cathode, negative to anode

2. size of molecules - move at different rates

3. 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

1. create agarose gel plate and add wells in one end

2. submerge gel in an electrolyte solution

3. load DNA fragments with micropipette

4. apply electric current

5. not visible yet - transfer to absorbent platter and head to separate 2 DNA strands

6. 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

1. target RNA/DNA sequence (whole genome not required) - section identified by primer sequence

2. DNA polymerase Taq Polymerase because it doesn’t denature at high temperatures

3. Free nucleotides

4. 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

  1. denaturation . heated to 95, breaks hydrogen bonds

  2. annealing - decreased to 50-60 so primer anneals to ends of single strands

  3. 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