Enzymes that act as

chemical scissors to
cut backbone of DNA
between deoxyribose
sugar and phosphate
groups at or near
specific recognition
sites.
Majority have been
isolated from bacteria

Importance:

A tool to produce DNA fragments of varying

size to be able to sequence the DNA.
It serves as host- defense mechanism of host
bacteria against viruses.

Most recognition sequences

are palindromes .
They read the same
forward (5' to 3' on the
top strand) and backward
(5' to 3' on the bottom
strand).

5' overhangs: The enzyme

cuts asymmetrically within
the recognition site such
that a short single-stranded
segment extends from the
5' ends.

3' overhangs: asymmetrical

cutting within the
recognition site, but the
result is a single-stranded
overhang from the two 3'
ends.
The 5' or 3' overhangs are
called sticky ends
or cohesive ends, because
they will readily stick or
anneal with their partner by
base pairing.

Blunts: Enzymes that

cut at precisely
opposite sites in the
two strands of DNA
generate blunt ends
without overhangs.

commonly used plasmid

cloning vector in E. coli .
a double-stranded circle 4,361
base pairs in length.
p stands for "plasmid," and BR
for "Bolivar" and "Rodriguez.
contains the genes for
resistance to ampicillin and
tetracycline
contains unique restriction
sites for several restriction
enzymes.

A method used to
separate and measure
the molecular size of
DNA and RNA
molecules using
electric field.
The migration rates of
DNA molecules are
inversely proportional
to their molecular
weights.

Negatively charged DNA

molecules are separated on
agarose gel matrix
according to their molecular
weight by applying an
electric field to move
towards the positive
electrode.
The position of DNA in the
agarose gel is visualized by
staining the gel with a
fluorescent intercalating
dye like Ethidium bromide.

A set known DNA fragments

with different sizes in base
pairs or kilo base pairs
which are separated as
bands on a gel like ladder.
They are used as
logarithmic scale by which
to estimate the size of the
other fragments.

Determine the size of an

unknown DNA fragment
by comparing it with DNA
ladders of known size.
Rf value: retention factor;
relative mobility

Measures the distance

traveled by each of the bands
of DNA ladder and test
samples from the well

The Rf of the DNA ladder

depends upon the log of
its relative molecular
weight.

Rf = Distance traveled by DNA

molecule
Distance traveled by the
dye

A standard curve
can be obtained by
plotting the
molecular size of
the fragments of
the marker against
the their respective
mobility.
The molecular size
of the test sample
can be obtained
from the standard
curve.