Transcript Southern Blotting

Southern Blotting
By
Erin Lucero
Dr. Adema
Bio 446
Who discovered Southern Blotting?
• Sir Edwin Mellow Southern discovered Southern
Blotting in 1975.
• At the time of the discovery he was working at
Medical Research Council Mammalian research
unit at Edinburg
• Founder, Chairman, and Chief scientific officer of
Oxford gene technology.
• Emeritus professor of biochemistry at the
University of Oxford.
• Along with discovering Southern Blotting, his
company developed the DNA microarray.
No Sequencing or PCR!
PCR
machine
• Before Sequencing or PCR individuals
relied on Southern blotting to detect
genes through out the entire genome.
• Can use Southern Blotting to detect
genetic markers such as, Restriction
Fragment Length Polymorphism.
• Blotting pattern is characteristic to a
specific genotype at a specific locus.
• Can use this technique to detect diseases
Next Generation Illumina
Sequencing machine
Methodology of Southern Blotting
• 32 P Isotope
•
The exact sequence of the probe was unknown
•
The DNA probe was specific to a certain region
of DNA, usually isolated from a specific
chromosome.
•
Hybridization between complementary DNA
and Probe.
•
Denature the ddDNA into ssDNA with PH, then load it
into gel.
Southern Blotting
Edmund Southern graphical representation of Southern Blotting in 1975. The manuscript was rejected by
Journal of Molecular Biology, E. Southern, "Detection of specific sequences among DNA fragments separated
by gel-electrophoresis," J Mol Biol, 98:503, 1975. (Cited in 30,666 papers)”
Restriction Fragment length polymorphisms
(RFLP)
• Polymorphism: is a genetic variant that appears in at least 1% of a population
• Such variations include: ABO blood type, Rhesus factor, and major
histocompatibility complex (MHC).
• RFLP are differences in homologous DNA sequences that can be detected by the
presence of fragments of different lengths after digestion of the DNA.
• Moreover, RFLP is a sequence of DNA that has a restriction site on each end with
a "target" sequence in between. A target sequence is any segment of DNA that
bind to a probe by forming complementary base pair.
• RFLP can be used to detect certain deleterious diseases, such as, sickle cell
anemia, and cystic fibrosis.
• The DNA is polymorphic among certain sites.
• When a single nucleotide changes in gene it is denoted as Single Nucleotide
polymorphisms.
Clinical Aspects of RFLP
• Genome mapping
• Paternity testing
• Genetic disorders
• DNA fingerprinting
RFLP Analysis
References
• https://en.wikipedia.org/wiki/Edwin_Southern
• https://en.wikipedia.org/wiki/Restriction_fragment_length_polymorphism
• https://employees.csbsju.edu/hjakubowski/classes/ch331/dna/NA_4C1_Lang_DNA.html
• http://www.the-scientist.com/?articles.view/articleNo/15193/title/The-Birth-of-theSouthern-Blot--1975/
• http://www.biology-pages.info/R/RFLPs.html
• https://www.britannica.com/science/polymorphism-biology
• ttp://www.chem.fsu.edu/chemlab/bch4053l/Medical%20Biochem/sequence/backgroun
d.html
• http://www.news-medical.net/life-sciences/Restriction-Fragment-Length-Polymorphism(RFLP)-Technique.aspx