DNA replication

DNA replication

• synthesis of new strands of DNA with the exact same base sequences as original strands

• required for:

  • growth

  • replacement of damaged tissues

  • reproduction to provide cells that develop into gametes

semi-conservative replication

• begins w/ separation of a parent DNA molecule into 2 single strands by breaking H bonds between bases

• each single strand used as a template for assembly of a replica

• semi-conservative bc includes one new strand and one conserved from parent molecule

• keeping one og strand and complimentary base pairing ensures genetic continuity with high degree of accuracy between generations

role of helicase

• unwinds double helix and separates the 2 strands by breaking hydrogen bonds between base pairs

role of DNA polymerase

• links nucleotides together to form new strands using parent strand as template

• each nucleotide in the new strand has the base that is complementary to the base of the nucleotide on the template strand

• new strands assembled on each template strand are identical in base sequence to the other strand

gel electrophoresis

• separates mixtures of charged macromolecules using an electric current

• electrodes placed at both ends of gel, DNA moves towards anode, wells into which DNA is loaded are at the cathode

• gel restricts movement, small molecules move faster than larger ones

• gel electrophoresis therefore separates DNA on the basis of size of molecule

PCR (polymerase chain reaction)

• used for copying DNA artificially

• needed:

  • DNA sample

  • taq DNA polymerase: heat stable DNA polymerase

  • primers: short DNA strands that bind to DNA in the sample after it has been split into single strands by heat. primers bind at the point where DNA polymerase should bind and start copying. 2 primers needed one for each single strand

  • DNA nucleotides: for assembling new strands

• temperature cycles:

  • 95 celsius to separate the 2 strands

  • cooled to 53 to allow primers to bind

  • increased to 73, encouraging taq polymerase to begin replicating from the primer onwards, producing 2 copies of double stra

applications of PCR and gel electrophoresis

• forensic investigations: evidence

• paternity tests: all bands in childs profile will also be in the profile of the mother or true father

DNA profiling process

• DNA sample collected from saliva/mouth

• PCR used to make copies of DNA

• short tandem repeats used

• gel electrophoresis used to separate DNA fragments according to length, generating pattern of bands

• all bands in child’s profile must be in one of parent’s profiles

directionality of DNA polymerase

• replication must occur in the 5’ to 3’ direction, adding nucleotides to the 3’ end of a single strand

replication on leading strand

• made continuously, following the replication fork as it opens

• RNA primer is assembled at the start of the leading strand to create binding point for DNA polymerase

• DNA polymerase can then assemble any length of new strand

replication on the lagging strand

• made discontinuously in short fragments, away from the replication fork

• DNA polymerase adds nucleotides to growing chain but soon reaches previous RNA primer beyond which new strand of DNA already assembled

• replication has to be reinitiated close to the replication fork with another RNA primer

• series of short lengths of DNA assembled on the lagging strand - okazaki fragments

function of enzymes in replication

1. helicase: unwinds double helix

2. DNA primase: adds a primer (short length of RNA)

3. DNA polymerase III binds to lagging strand at 3’ end of RNA primer and adds DNA nucleotides away from replication fork

4. DNA polymerase I removes RNA primer and replaces it with nucleotide but leaves a nick

5. DNA ligase seal nick by forming sugar phosphate bond and joins okazaki fragments together

6. DNA primase adds primer to 3’ end of leading strand

7. DNA polymerase III binds and assembles DNA towards replication fork continuously

correcting base mismatches

• when DNA polymerase III recognises a base mismatch, it excises the incorrect nucleotides, moves back along template strand and re-inserts correct base pair - this is DNA proofreading