D1.

1 DNA replication
Syeda Farah Husyn
Semi-conservative nature of DNA replication
Polymerase Chain Reaction (PCR)
Gel electrophoresis
• Gel electrophoresis is a process that is used to separate molecules such as
proteins and fragments of nucleic acids.
• ln electrophoresis, proteins or nucleic acid fragments are separated on the basis
of their overall charge and mass. Separation is due to the following factors:
Differential migration of these molecules through a supporting medium - typically
this is either agarose gel or polyacrylamide gel (PAG). ln these media, the tiny
pores in the gel act as a molecular sieve; small particles can move quite quickly,
whereas larger molecules move much more slowly.
The electrical charge that molecules carry - it ls the phosphate groups in DNA
fragments that give them a net negative charge. Consequently, when these
molecules are placed in an electric field, they migrate towards the positive pole
(anode) .
Applications of PCR
and gel electrophoresis
• DNA sequencing
• Genetic fingerprinting: a laboratory
technique used to determine the probable
identity of a person based on the nucleotide
sequences of certain regions of human DNA that
are unique to individuals.
Identification of suspects
Identification of corpses
Determining paternity
AHL
Directionality of DNA polymerases
• DNA polymerase requires a free 3'-0H group to start the synthesis of a
new DNA strand, adding the 5' of a DNA nucleotide to the 3' end of a
strand of nucleotides. This means it can synthesize in only one
direction, moving along the template strand in a 3' to 5' direction, and
synthesizing the new DNA strand in a 5' to 3' direction.
Enzymes in DNA replication
• Replication is be initiated by an RNA primer.
• Semi-conservative replication is actually initiated at many points along
the DNA double helix. These points are known as replication forks
• DNA strands separate brought about by the enzyme helicase.
• Another enzyme, DNA gyrase, untwists the DNA
• Then, single-strand binding proteins attach and prevent the
separated strands from rejoining.
• DNA polymerase lll: the primary enzyme involved in DNA replication
https://youtu.be/TNKWgcFPHqw?si=PsrSB7dySvGFOAXL
DNA proofreading