DNA REPAIR MECHANISMS

Photoreactivation

Excision Repair
a. Base Excision Repair

b. Nucleotide Excision
Repair

Enzymes involved in Neutralization

of Free Radicals
1. Superoxide dismutase (SOD)
2H+ O2 + H2O2
2. Catalase
2H2O2 O2 + H2O
3. Glutathione Peroxidase
2OH + 2GSH 2H2O + GSSG

2O2 +

Vitamins

Recombination Repair

Demethylase
SH + G-CH3

Sulfitase
HSO3- SO4-2

S-CH3

+ G

Nucleic Acid Biotechnology

What is Recombinant DNA?

DNA molecule containing DNA fragments (from

different sources) that are not naturally found.

CHIMAERA
A mythical creature with the head of a lion, the body of a goat, and the
tail of a serpent

Gene Transfer

Restriction Endonucleases?!
Restriction endonucleases
recognize and cut specific
sequences in the doublestranded DNA.
This specific base sequence is
known as the "recognition
sequence"

STICKY Ends

VECTORS

Vectors - gene carrier;

also known as
cloning vehicle

VECTOR CHARACTERISTICS

It must be small, and unlikely to

degrade during purification
It has markers (such as antibiotic
resistance) that can indicate (in
culture) whether transformation
has been successful
It must have an origin of
replication so that DNA can be
replicated by the host cell's
machinery
It has have several unique
restriction sites so that the vector
DNA will be cut only in the desired
location, and that several such
locations will be available for
insertion of foreign DNA

pbr322
4361bp

(SMORES!)

Genetic
Engineering:
Recombinant DNA
Technology and
Gene Cloning

Recombinant DNA
Technology
Steps:
1. Isolation of DNA
2. Cleaving of DNA with
restriction enzymes
3. Formation of recombinant
DNA
4. Introduce recombinant
DNA to host cells
5. Propagation of clones

Genetic Engineering

Polymerase Chain Reaction

It is first developed by Kary
Mullis in 1983
A more convenient method
of amplifying specific DNA
fragment.
Allows investigation of
minute samples of DNA

PCR: in vitro repliaction

It allows you to carry
out in vitro multiple
replications of target
DNA
Can produce millions of
copies DNA fragments
from a single template
PCR is very specific

Components of PCR tubes

DNA template, which contains the
region of the DNA fragment to be
amplified
Two primers, which determine the
beginning and end of the region to be
amplified
DNA polymerase, which copies the
region to be amplified
Nucleotides (dNTPs), from which the
DNA polymerase builds the new DNA
Buffer, which provides a suitable
chemical environment for the DNA
polymerase

Instrument for PCR

The PCR reaction is carried out in a thermocycler, an
instrument that automatically controls and alternates the
temperatures for programmed periods of time for the
appropriate number of PCR cycles (usually bet 30 - 40
cycles)

Three Steps in PCR

There are three major

steps in a PCR, all carried
out in the same test tube
but at different
temperatures:
1. Denaturation at >90C
2. Annealing at 54C
3. Extension at 72C

2. ANNEALING
3. EXTENSION

1. DENATURATION

PCR

Exponential Amplication

Applications of PCR
For rapid diagnosis of infectious diseases
Detection of rare pathological events such as
mutation leading to cancer
Examine DNA of extinct species (mammoth,
dinosaurs)
In criminal investigations, DNA fingerprints are
prepared from the cells in a tiny speck of dried
blood or semen or at the base of a single human
hair
Physicians can detect genetic defects in very early
embryos by collecting a sloughed off cells and
amplying DNA

Paternity Testing/Forensics