Southern Blot.docx

BIO198L Gene Biotechnology Laboratory 1st Quarter SY 2015-2016

Southern Blot Lomugdang, Fiord Jogardy B.1 1Student

(s), Subject/Section, School of Chemical Engineering, Chemistry and Biotechnology, Mapua Institute of Technology

INTRODUCTION Southern blotting is one of the central techniques in molecular biology. First devised by E. M. Southern (1975) ,Southern blotting results in transfer of DNA molecules, usually restriction fragments, from an electrophoresis gel to a nitrocellulose or nylon sheet (referred to as a ‘membrane’) , in such a way that the DNA banding pattern present in the gel is reproduced on the membrane. During transfer or as a result of subsequent treatment, the DNA becomes immobilized on the membrane and can be used as a substrate for hybridization analysis with labelled DNA or RNA probes that specifically target individual restriction fragments in the blotted DNA. In essence, Southern blotting is therefore a method for ‘detection of a specific restriction fragment against a background of many other restriction fragments’. The restricted DNA might be a plasmid or bacteriophage clone, Southern blotting being used to confirm the identity of a cloned fragment or to identify an interesting sub fragment from within the cloned DNA, or it might be genomic DNA, in which case Southern blotting is a prelude to techniques such as restriction fragment length polymorphism (RFLP) analysis.

MATERIALS AND METHODS The following were the methods were used in this experiment: I. Choose cut DNAs to fill the lanes in the gel II. Cross link DNA to the filter. III. Set up the prehybridization. IV. Select a radioactively labelled probe. V. Denature the probe. VI. Add the probe to the roller bottle. VII. Wash the filter. VIII. Place the filter in the X-ray cassette.

The objectives of this experiment are to choose genomic DNAs for the Southern blot, select a fragment of DNA to use as a probe, hybridize the Southern Blot with the probe and interpret the hybridization pattern.

RESULTS & DISCUSSION

Figure 1. Southern blotting.

Experiment 06│ Date: September 04, 2015

In the experiment, genomic DNAs were chosen to fill the lanes in the gel. There were six genomic DNAs that were chosen. Then, cross-linking pf the DNA to the filter were performed. The permanent attachment of DNA to the nylon filter requires exposing it to ultraviolet (UV) light. During gel electrophoresis, the DNA is bound by ethidium bromide. When exposed to UV light, ethidium

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BIO198L Gene Biotechnology Laboratory 1st Quarter SY 2015-2016

bromide emits an orange color, making RNA visible on the nylon filter. Next, pre-hybridization was performed. Hybridization analysis is based on the principle that two polynucleotides will form a stable hybrid by base-pairing if their nucleotide sequences are wholly or partly complementary. A specific restriction fragment in a Southern blot can therefore be detected if the membrane is probed with a second, labelled DNA molecule that has the same, or similar, sequence as the fragment being sought. A probe that hybridizes only to a single DNA segment that has not been cut by the restriction enzyme will produce a single band on a Southern blot, whereas multiple bands will likely be observed when the probe hybridizes to several highly similar sequences (e.g., those that may be the result of sequence duplication). Modification of the hybridization conditions for instance, increasing the hybridization temperature or decreasing salt concentration, may be used to increase specificity and decrease hybridization of the probe to sequences that are less than 100% similar.

Figure 3. X-ray image.

As seen from the figures above, figures 1 and 2, all sample DNA were cut by the same restriction enzymes KpnI and AccI. The DNA sequence for the β-globin is in band 4.5 which is 1400 bp long. The DNA of the normal person, sickle cell anaemia patient, βthalassemia patients 2, 3 and 4 showed same band at 4.5 cm while β-thalassemia patient 1 showed no sign of the band. This must be the result of the restriction enzymes detecting a restriction site in the DNA sequence of β-thalassemia patient number 1. The probe used in this experiment was the β-globin probe which detects the sequence for the β-globin gene. The experiment showed that βthalassemia patient had the gene that contained a restriction site for the restriction enzymes KpnI and AccI. This indicates that the gene had a deletion mutation.

Figure 2. Ultraviolet image

Experiment 06│ Date: September 04, 2015

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BIO198L Gene Biotechnology Laboratory 1st Quarter SY 2015-2016

CONCLUSION Southern blotting is a technique that enables a specific restriction fragment to be detected against a background of many other restriction fragments. It involves transfer of DNA fragments from an electrophoresis gel to a nitro-cellulose or nylon membrane in such a way that the DNA banding pattern present in the gel is reproduced on the membrane. Hybridization probing is then used to detect the restriction fragment that is being sought. The basic methodology for Southern blotting has not changed since the original technique was described in 1975, but modifications have been introduced with the aim of speeding up the process and achieving a more efficient transfer. Southern blotting has many applications in molecular biology, including the identification of one or more restriction fragments that contain a gene or other DNA sequence of interest and in the detection of RFLPs used in construction of genomic maps. REFERENCES Moffatt, B (2006). Course Notes Biology 208.Waterloo, University of Waterloo. Dale, J, & von Schantz, M (2003). From Genes to Genomes.West Sussex: John Wiley & Sons Ltd. C. Starr - R. Taggart: Cell Biology and Genetics, Brooks/ColeThomson Learning, 2004. Robert F. Weaver.—5th ed. Molecular Biology, Published by McGraw-Hill, 2012.

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