Southern

Blotting

Nurhidayatullah
Ingratsusi Marviani

Yang akan di Bahas

Pendahuluan

1. Apa itu Blotting?

Blots are techniques for transferring DNA , RNA and

proteins onto a carrier so they can be separated, and
often follows the use of a gel electrophoresis. The
Southern blot is used for transferring DNA, the
Northern blot for RNA and the western blot for
PROTEIN.

TYPES OF BLOTTING TECHNIQUES

Blotting
technique

Southern Blot

Northern Blot

Western blot

It is used to
detect DNA.

It is used to
detect RNA.

It is used to
detect protein.

Southern Blotting

Professor Sir Edwin Southern, Professor of

Biochemistry and Fellow of Trinity developed this
method in 1975.
Southern won the Lasker Award for Clinical Medical
Research prize for the method of finding specific
DNA sequences he developed this procedure at
Edinburgh University more than 30 years ago. The
technique is known as DNA transfer or 'Southern
blotting'

Southern Blotting

Teknik ini digunakan untuk pendeteksian suatu

DNA sequence spesifik (gen atau lain) dalam
sample kompleks DNA (selular DNA). Ini juga
digunakan untuk menentukan bobot molekular
suatu restriksi fragmen dan untuk mengukur
sejumlah relatif dalam sample berbeda.

Tahapan Southern
Blotting

1. Digest the DNA with an appropriate

restriction enzyme.

Tahapan Southern
Blotting
2.The complex mixture of fragments is subjected to gel
electrophoresis to separate the fragments according to
size.

Tahapan Southern
Blotting

3. The restriction fragments present in the gel are

denatured with alkali and transferred onto

Tahapan Southern
Blotting
4. a nitrocellulose filter or nylon membrane by
blotting.

Tahapan Southern
Blotting
5.The filter is incubated under hybridization conditions
with a specific radio labeled DNA probe.

The probe hybridizes

to the complementary
DNA restriction
fragment.

Tahapan Southern
Blotting
6.Excess probe is washed away and the probe bound to the filter is
detected by autoradiography, which reveals the DNA fragment to
which the probe hybridized.

Tahapan Southern
Blotting

Aplikasi Metode Southern

Blotting

Southern blots are used in gene discovery ,

mapping, evolution and development studies,
diagnostics and forensics (It is used for DNA
fingerprinting, preparation of RFLP maps)
identification of the transferred genes in
transgenic individuals, etc.

DNA Fingerprint
DNA fragments show unique patterns from
one person to the next.
Used in paternity disputes and as forensic
evidence.

Preparing a DNA Fingerprint

Specimen Collection- Could be a licked envelope,
dirty laundry, a cigarette butt, saliva

Special precautions in handling specimens: gloves,

disposable instruments, avoid talking and sneezing, avoid
touching sample with your skin, air-dry the evidence
before packaging so mold does not grow
Enemies of evidence: sunlight, high temperatures,
bacteria, moisture
Ideal sample: 1 mL of fresh, whole blood (white blood
cells) treated with EDTA

RFLP
Restriction Fragment Length Polymorphism
(RFLP)

Nucleotide sequence variations in a region of DNA

that generates fragment length differences
according to the presence or absence of restriction
enzyme recognition sites.

RFLP
The RFLP fragments can be separated by gel
electrophoresis.

RFLP

Southern Blot

Southern Blot Animation