Editing Southern blot (section)

== Method ==
The genomic DNA is digested with either one or more than one restriction enzyme, then the DNA fragments are size-fractionated by gel electrophoresis. Before the DNA fragments are transferred to a solid membrane which is either nylon or nitrocellulose membrane they are first denatured by alkaline treatment.<ref name=":0" /> After the DNA fragments are immobilized on the membrane, prehybridization methods are used to reduce non-specific probe binding. Then the fragments on the membrane are hybridized with either radiolabeled or nonradioactive labeled DNA, RNA, or oligonucleotide probes that are complementary to the target DNA sequence. Then detection methods are used to visualize the target DNA.<ref name=":2">{{Cite journal |last1=Green |first1=Michael R. |last2=Sambrook |first2=Joseph |date=July 2021 |title=Analysis of DNA by Southern Blotting |journal=Cold Spring Harbor Protocols |language=en |volume=2021 |issue=7 |pages=pdb.top100396 |doi=10.1101/pdb.top100396 |pmid=34210774 |s2cid=235710916 |issn=1940-3402|doi-access=free }}</ref> 
#'''DNA Isolation:''' The DNA to be studied is isolated from various tissues. The most suitable source of DNA is known as blood tissue. However, it can be isolated from different tissues (hair, semen, saliva, etc.).
#'''DNA digestion:''' Restriction [[endonuclease]]s are used to cut high-molecular-weight DNA strands into smaller fragments. This is done by adding the desired amount of DNA which can be changed according to the probe used and the intricacy of the DNA, with the restriction enzyme, enzyme buffer and purified water. Then everything is incubated at 37&nbsp;°C overnight.
#'''Gel electrophoresis:''' The DNA fragments are then [[Gel electrophoresis|electrophoresed]] on an [[Agarose gel electrophoresis|agarose gel]] to separate them by size. If some of the DNA fragments are larger than 15 [[Base pair#Length measurements|kb]], then before blotting, the gel may be treated with an acid, such as dilute [[Hydrochloric acid|HCl]]. This [[Depurination|depurinate]]s the DNA fragments, breaking the DNA into smaller pieces, thereby allowing more efficient transfer from the gel to membrane.
# '''Denaturation:''' If alkaline transfer methods are used, the DNA gel is placed into an alkaline solution (typically containing [[sodium hydroxide]]) to denature the double-stranded DNA. The denaturation in an alkaline environment may improve binding of the negatively charged thymine residues of DNA to a positively charged amino groups of membrane, separating it into single DNA strands for later [[Nucleic acid hybridization|hybridization]] to the probe (see below), and destroys any residual RNA that may still be present in the DNA. The choice of alkaline over neutral transfer methods, however, is often empirical and may result in equivalent results.{{Citation needed|date=February 2009}}
# '''Blotting:''' A sheet of [[nitrocellulose]] (or, alternatively, [[nylon]]) [[artificial membrane|membrane]] is placed on top of (or below, depending on the direction of the transfer) the gel.  Pressure is applied constantly to the gel (either using suction, or by placing a stack of paper towels and a weight on top of the membrane and gel), to ensure good and even contact between gel and membrane. If transferring by suction, [[SSC buffer|20X SSC]] buffer is used to ensure a seal and prevent drying of the gel. Buffer transfer by [[capillary action]] from a region of high [[water potential]] to a region of low water potential (usually filter paper and paper tissues) is then used to move the DNA from the gel onto the membrane; [[ion exchange]] interactions bind the DNA to the membrane due to the negative charge of the DNA and positive charge of the membrane. Five methods can be used to transfer DNA fragments to the solid membrane and they are:<ref name=":2" />  
##Upward capillary transfer: This method transfers the DNA fragment upward from the gel to the membrane where the flow of the liquid or the buffer will be upward. 
##Downward capillary transfer: This method is done by placing the gel on the surface of the membrane (usually nylon charged membrane) and the DNA fragments will be transferred in a downward direction with the flow of the alkaline buffer. 
##Simultaneous transfer to two membranes: This method is used to transfer DNA fragments of high concentration simultaneously from the gel to two membranes. 
##Electrophoretic transfer: This method usually uses large electric current which makes it difficult to transfer the DNA efficiently due to the temperature of the buffer used, so these machines can be either equipped with cooling machines or used in a cold area.
##Vacuum transfer: This method uses a buffer from the upper chamber to transfer the DNA from the gel to the nitrocellulose or nylon membrane, the gel is placed directly on the membrane, and the membrane is placed on a porous screen on the vacuum chamber.
# '''Immobilization:''' The membrane is then baked in a vacuum or regular oven at 80&nbsp;°C for 2 hours (standard conditions; nitrocellulose or nylon membrane) or exposed to [[ultraviolet radiation]] (nylon membrane) to permanently attach the transferred DNA to the membrane.
# '''Hybridization:''' After that, a [[hybridization probe]]—a single DNA fragment with a particular sequence whose presence in the target DNA is to be ascertained—is exposed to the membrane. The probe DNA is labelled so that it can be detected, usually by incorporating [[radioactivity]] or tagging the molecule with a [[fluorescence|fluorescent]] or [[Chromogenic in situ hybridization|chromogenic dye]].  In some cases, the hybridization probe may be made from RNA, rather than DNA. To ensure the specificity of the binding of the probe to the sample DNA, most common hybridization methods use salmon or herring sperm DNA for blocking of the membrane surface and target DNA, deionized [[formamide]], and detergents such as [[sodium dodecyl sulfate|SDS]] to reduce non-specific binding of the probe.
# '''Detection:''' After hybridization, excess probe is washed from the membrane (typically using [[SSC buffer]]), and the pattern of hybridization is visualized on [[X-ray]] film by [[autoradiography]] in the case of a radioactive or fluorescent probe, or by development of color on the membrane if a chromogenic detection method is used.