University:
Cameron University
Course:
1004 | General Biology
Academic year:

2019
Views:

98

Pages:

1
Author:

Clint W.

DNA REPLICATION Semi-Conservative DNA replication is semi-conservative – one strand is from an original template molecule and one strand is newly synthesised • This occurs because each base will only pair with its complementary partner and thus ensure the sequence is conserved Original strand Newly synthesised DNA Replication Helicase: • Unwinds and separates the double stranded DNA • Breaks the hydrogen bonds between the base pairs DNA Polymerase III • Free nucleotides line up opposite complementary partners • DNA Pol III covalently joins the free nucleotides together 5’ → 3’ DNA Pol III Helicase 3’ ← 5’ Meselson-Stahl Experiment Polymerase Chain Reaction The Meselson-Stahl experiment supported the theory that DNA replication occurred via a semi-conservative process The polymerase chain reaction (PCR) is an artificial method of DNA replication that is used to rapidly copy sequences They incorporated radioactive nitrogen isotopes into DNA • Templates were prepared with heavier 15N • New sequences were replicated with lighter 14N PCR occurs in a thermal cycler over three repeating steps: • Denaturation: DNA heated in order to separate strands • Annealing: Primers attach to ends of a target sequence • Elongation: A heat-tolerant polymerase copies strands The DNA was then separated via centrifugation in order to determine its composition of radioisotopes • 1st division: DNA had 15N and 14N (i.e. mixed) • 2nd division: DNA is mixed or has 14N only The results were consistent with a semi-conservative model 2nd Division 1st Division ACTUAL RESULT SEMI-CONSERVATIVE A standard reaction of 30 cycles would generate 230 copies of the target DNA sequence (i.e. >1 billion copies of DNA) 1. Denaturing Strands separate (95°C) 2. Annealing N-15 / N-14 N-15 Mix (×2) Primers bind (55°C) 3. Extension N-14 only N-15 / N-14 Mix Mix N-14 Taq polymerase (72°C)