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Nurhidayatullah Ingratsusi MarvianiSouthern BlottingYang akan di BahasPendahuluan1. 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 TECHNIQUESBlotting technique

Southern Blot

It is used to detect DNA.

Northern Blot

It is used to detect RNA. Western blot

It is used to detect protein.Southern BlottingProfessor 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'

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.

Southern BlottingTahapan Southern Blotting1. Digest the DNA with an appropriate restriction enzyme.

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

Tahapan Southern Blotting3. The restriction fragments present in the gel are denatured with alkali and transferred onto

Tahapan Southern Blotting4. a nitrocellulose filter or nylon membrane by blotting.

Tahapan Southern Blotting5.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 Blotting6.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

Tahapan Southern BlottingAplikasi Metode Southern BlottingSouthern 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 FingerprintDNA fragments show unique patterns from one person to the next.Used in paternity disputes and as forensic evidence.

A little over a hundred years ago scientists discovered that patterns in the skin of the fingertips could be used to establish identity. In 1985 scientific researchers discovered a new kind of fingerprint. The unique signature in every persons genetic makeup is called a DNA fingerprint. The uniform nature of DNA in a single individual and the genetic variability between individuals make DNA fingerprints useful to establish paternity and can also be used to implicate or exonerate a suspect in a crime.Preparing a DNA FingerprintSpecimen Collection- Could be a licked envelope, dirty laundry, a cigarette butt, salivaSpecial 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, moistureIdeal sample: 1 mL of fresh, whole blood (white blood cells) treated with EDTARFLPRestriction 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.

Restriction Fragment Length Polymorphisms, or RFLPs for short, are generated due to mutations in recognition sites. Remember what a recognition site is? Correct! Its the specific nucleotide sequence recognized by restriction enzymes. The restriction enzymes bind to the DNA and cut within the recognition site. If the nucleotide sequence has been mutated, then the restriction enzyme will not bind and therefore will not cut at that site. As this example shows, the change in the recognition site will produce different length fragments in a restriction digest. Individual one has two recognition sites for the EcoRI enzyme. That means EcoRI will cut this DNA fragment twice. What happens when you cut a piece of string twice? You get three pieces. The same is true when you cut linear DNA with molecular scissors like restriction enzymes. Two cuts generates three fragments labeled here as A, B, and C. In individual 2 the first recognition site is not present. Instead of GAATTC the sequence is now GAAATTC. EcoRI will not bind and cut at this location. That means in the same restriction digest, the restriction enzyme will only cut once and generate two fragments, D and C.RFLPThe RFLP fragments can be separated by gel electrophoresis.

The fragments from the restriction digests can be separated by gel electrophoresis. Because the fragments are different lengths, they will run to different locations on a gel. This creates the unique RFLP pattern, or DNA fingerprint, for each individual.RFLP

Click on the RFLP animation to visualize the creation of a unique DNA fingerprint.Southern Blot AnimationSouthern Blot

Click on the Southern Blot Animation to visualize this process.