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SOUTHERN BLOTTING
Nimmy Francis S2; M.Sc Microbiology

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NUCLEIC ACID HYBRIDIZATION

Nucleic acid hybridization includes a variety of related techniques that are based on the observation that two single-stranded nucleic acid molecules of complementary base sequence can form a double-stranded hybrid under proper conditions.
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In Complementary Sequence Analysis

Complementary sequences - similarity in nucleotide sequence between two samples of DNA, from two different organisms. The more distant the evolutionary relationship between the two species, the greater the divergence of their DNA sequences.
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When DNAs from different species are allowed to reanneal in different combinations, the melting

temperature (Tm,) of the hybrid duplexes provides a measure of the

evolutionary organisms. distance between the

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PROBES

A DNA molecule of defined oligonucleotide sequence either a purified fragment or a chemically synthesized To search mixtures of nucleic acids for complementary sequences Radioactive labeled probesP32/S35 incorporated into the alpha- phosphate of one of the 66

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Methods of radioactive labeling are

Radioactive Labeling

PCR technique

*d NTP and DNA Polymerase

Labeling the end of intact oligonucleotide

Polynucleotide kinase + ATP 5 OH will be phosphorylated with 77 P

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Stringent Vs. Non-stringent Hybridization

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BLOTTING TECHNIQUES
Gel Transfer Hybridization Technique

SOUTHERN BLOTTING - DNA NORTHERN BLOTTING - RNA WESTERN BLOTTING IMMUNOBLOTTING - Protein - Antibody
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SOUTHERN BLOTTING

Dr. Edwin Southern Slower and more involved than direct visualization by fluorescent dye technique

Highly sensitive

Allow the specific detection of defined sequences of interest among many similarly sized bands on a gel 4/28/12 1414

Determining the Location of Specific DNA Fragments in a Gel by a Southern Blot.

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Restriction Nucleases Produce DNA Fragments

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PH O TR IS EC ES EL

R O

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Preparation of Gels for Blotting

Fragmentation of large DNA molecules (optional)

DNA fragments longer than 10 kb do not transfer to blotting membranes efficiently. In order to facilitate their transfer, these fragments are reduced in size, either by acid depurination or by UV irradiation.

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Acid depurination immediately after gel electrophoresis, place the gel in a solution of 0.2 M HCl, so that it is completely covered. During this period the color of the bromophenol blue in the samples will change from blue to yellow, indicating that the gel has been completely saturated with the acid.

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Depurinated gels may yield fuzzy bands on the final autoradiograph, presumably because of increased diffusion of the DNA during transfer. Depurination is therefore recommended only when fragmentss larger than 10 kb are to be transferred. UV irradiation expose the gel to UV light at a wavelength of 254 nm from a source operating at 30 W, for 3060 seconds.

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Denaturation/ Alkylation

Double-stranded DNA must be denatured in order to create suitable hybridization targets. Completely cover the gel with denaturation buffer and incubate for 30 minutes with gentle shaking. If acid depurination was used to denature the DNA, the bromophenol blue will return to its original color during this incubation. 4/28/12 2121

BLOTTIN G

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FIXING

Fix the DNA to the blot, either by baking or by UV-crosslinking To fix the DNA to the membrane by baking, first let the blot airdry on a sheet of filter paper, then place between two sheets of filter paper, and bake at 80C for 2 h. To fix the DNA by UV-crosslinking, first protect the surface of the membrane by covering the UV source (e.g., a transilluminator) with 4/28/12 2323

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Blocking Agents
Blocking agents prevent ligands from sticking to surfaces. They are used in molecular cloning to stop nonspecific binding of probes in Southern, northern, and western blotting. If left to their own devices, these probes would bind tightly and nonspecifically to the supporting nitrocellulose or nylon membrane. Without blocking agents, it would be 4/28/12 2525 impossible to detect anything but the

Blocking Agents Used for Nucleic Acid Hybridization

Two blocking agents in common use are Denhardt's reagent (Denhardt 1966) and BLOTTO (Bovine Lacto Transfer Technique Optimizer; Johnson et al. 1984). Usually, the filters carrying the immobilized target molecules are incubated with the blocking agents for an hour or two before the probe is 4/28/12 2626 added.

ADDITION OF PROBES

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Visualization

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DNA FINGERPRINTING

The technique is widely used in forensic laboratories to identify individuals who have left blood or other DNAcontaining tissues at the scenes of crimes. Alec Jeffrey and colleagues in 1985. his
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How do you figure out that someones DNA is more similar to anothers? The primary method of assessing similarities is by use of DNA fingerprinting or DNA restriction analysis.

This process makes use of special proteins called restriction enzymes and sections of the chromosome called tandem repeats 4/28/12 3030

Tandem Repeats

A region of the chromosome that contains multiple copies of a core DNA sequence that are arranged in a repeating fashion Repeats act as fillers or spacers between coded sections of DNA

All humans have the same type of repeats but there is tremendous variation in the number of repeats 4/28/12 3131 that each of us has hence known

Minisatellite DNAs

Minisatellite sequences range from about 10 to 100 base pairs in length and are found in sizeable clusters containing as many as 3000 repeats.

Minisatellites tend to be unstable, and the number of copies of a particular sequence often increases or decreases from one 4/28/12 3232

Restriction Fragment Length Polymorphisms (RFLP)

RFLPs are different fragment lengths of base pairs that result from cutting a DNA molecule with a restriction enzyme

It is the length differences associated with DNA strands or RFLPs that allow one to distinguish one person from another. 4/28/12 3333

PROTOCOL

A DNA fingerprint is really just a Southern blot. To make one, investigators first cut the DNA under study with a restriction enzyme such as HaeIII.

Jeffrey chose this enzyme because the repeated sequence he had found did not contain a HaeIII recognition site. 4/28/12 3434

The enzyme haeIII cuts the DNA in seven places (short arrows), generating eight fragments.

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Using RFLP

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Electrophor esis

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Paternity test

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DNA Typing

One advantage of DNA typing is its extreme sensitivity. Only a few drops of blood or semen are sufficient to perform a test. However, sometimes forensic scientists have even less to go ona hair pulled out by the victim, for example. Selected segments of DNA from 4/28/12 4040 these cells can be amplified by PCR

Practical Applications of DNA Fingerprinting

1.Paternity and Maternity

person inherits his or her VNTRs from his or her parents Parent-child VNTR pattern analysis has been used to solve standard father-identification cases
Can someone tell me who is my father?

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2. Criminal Identification and Forensics

DNA isolated from blood, hair, skin cells, or other genetic evidence left at the scene of a crime can be compared

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3. Personal Identification

The notion of using DNA fingerprints as a sort of genetic bar code to identify individuals has been discussed diagnose inherited disorders in both prenatal and newborn babies These disorders may include cystic fibrosis, hemophilia, Huntington's disease, familial Alzheimer's, sickle cell anemia, thalassemia, and many others.
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4.Diagnosis of Inherited Disorders

5.Developing Cures for Inherited Disorders

By studying the DNA fingerprints of relatives who have a history of some particular disorder identify DNA patterns associated with the disease

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Problems with Determining Probability

A. Population Genetics

VNTRs, because they are results of genetic inheritance it will vary depending on an individual's genetic background

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B. Technical Difficulties

Errors in the hybridization and probing process must also be figured into the probability Until recently, the standards for determining DNA fingerprinting matches, and for laboratory security and accuracy which would minimize error
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Thank You

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