Blotting is primarily used in molecular biology. It is used to identify proteins and nucleic acids for diagnostic purpose. Specifically, blotting is used for identifying biomolecules like DNA, mRNA, and protein during gene expression.
DNA and RNA molecules need to undergo biochemistry analysis and they are separated using the blotting method.
- Dot and slot blotting are simple techniques for immobilizing bulk unfractionated DNA on a nitrocellulose or nylon membrane. Hybridization analysis can then be carried out to determine the relative abundance of target sequences in the blotted DNA preparations. Dot and slot blots differ only in the geometry of the blot, a series of spots giving a hybridization pattern that is amenable to.
- DNA was denatured at 95 °C for 10 min prior blotting on to Hybond N+ (Amersham Pharmacia Biotech, England) membrane in a slot blot apparatus (Cleaver Scientific Co., England) under vacuum. The membrane was UV cross-linked (Church and Gilbert 1984) at 12 × 104 μJ/cm2 energy in a CL-1000 Ultraviolet crosslinker (UVP, Upland, CA, USA).
Southern and Northern blotting protocols involve the following major steps: Purification of DNA/RNA: Extract and purify the DNA/RNA from either cells or tissue sources. Digestion of DNA: Digest the DNA into fragments with restriction enzymes. This step is not required for RNA. Slot blot quantitation can help determine whether the DNA is human or non-human. DNA quantitation must be accomplished on all samples by yield gel and/or slot blot before amplification. Slot blot is required for all unknown samples. It is not required for standard samples. The decision to use slot blot on standard samples is left to the. Regularly it is useful to evaluate the abundance of certain RNA or DNA in the extracted nucleic acid mixture by dot blot or slot blot hybridisation without prior digestion and electrophoresis.
Picture : A helpful mnemonic for students to remember the relation between various blotting methods and its uses
Blotting is done by enabling the mixture of molecule pass through a gel that separates the molecules based on their molecular size. The molecules being tested are hard-pressed against a membrane which will then transfer the molecule from the gel onto the suitable membrane through capillary action.
One of the commonly used blotting procedures is Southern blotting. This procedure was introduced in 1975 by Edwin Southern; thus, where the name was taken. What is Southern blotting? It is a blotting method used to detect a particular sequence of DNA in a DNA sample. There are other subtypes such as Northern blotting, Western blotting, South-Western blotting, and Eastern blotting. (1, 2, 3, and 4)
Image 1: The Southern blot procedure as shown in the image above.
Picture Source: thescienceinfo.com
What does Southern blotting reveal?
A southern blot analysis reveals the following:
- Identity of the DNA
- Size of the DNA
- Abundance of the DNA (4, 5)
The Southern blotting method is a classic technique that separates DNA fragments according to their size through electrophoresis. They will be transferred to a membrane, hybridize, washed, and lastly, detecting the labeled DNA band.
How much DNA is needed for Southern blotting?
North america poker tour schedule. The amount of DNA needed varies depending on the size and specific activity of the probe.
What is the principle of Southern blotting?
Southern blotting is a restriction fragment length polymorphism. It is a hybridization method for identifying the size of DNA from a mixture of other similar molecules. Basically, Southern blotting separates DNA fragments by gel electrophoresis. The DNA fragments are identified using a labeled probe hybridization. (5, 6, and 7)
What are the steps in Southern blotting?
There are a series of steps that need to be strictly followed when performing Southern blotting. They are the following:
Image 2: The first and second step of southern blot method.
Picture Source: slideplayer.com
#1 – Restriction Digest
A restriction enzyme is used to fragmentize the DNA. The DNA is cut at a specific site generating a fragment. The DNA fragments obtained by restriction digest are amplified by PCR. (7, 8)
#2 – Gel electrophoresis
Once the DNA fragments are obtained, the next step is separating the DNA fragments using gel electrophoresis.
Image 3: The denaturing of DNA.
Picture Source: slidesharecdn.com
#3 – Denaturation
An SDS gel is needed in this step. Once electrophoresis is done, the SDS gel is soaked in either acid like HCl or alkali like NaOH. The purpose is to denature the fragments of double-stranded DNA. After denaturation, the strands of DNA will separate. (8, 9)
#4 – Blotting
This is where the actual bloating takes place. The DNA’s separated strands are transferred to a positively charged nylon membrane through the process of blotting.
Blotting process is done so as to determine the nature of DNA
#5 – Baking and Blocking
The DNA is based on autoclave and fix in the membrane. Casein or Bovine serum albumin is used to treat the membrane. What these chemicals do is they saturate the membrane’s binding site.
#6 – Hybridization with labeled probes
A labeled probe is used to treat the membrane-bound DNA. It has complementary sequences to the gene being studied on. The probe will bind with the complementary DNA on the membrane. (3, 7, and 10)
#7 – Visualization by Autoradiogram
An autoradiogram is used to visualize the membrane bound DNA that is labeled with a probe. Autoradiogram visualization provides a pattern of bands.
Why is Southern blotting important?
Image 5: A southern blot is one of the procedures used in paternity testing.
Picture Source: slideplayer.com
Southern blotting is important as it is useful in the various field of interest such as:
- It is used in detecting the DNA in a given sample such as DNA fingerprinting in forensics.
- It is useful in isolating and identifying the gene of interest.
- In forensics, southern blotting is one of the commonly used procedures, especially in:
- Criminal identification
- Victim identification
- It is used for paternity testing.
- It helps identify gene rearrangement or mutation in a particular sequence of DNA/gene mapping.
- It is used to diagnose a particular disease caused by genetic defects.
- It is helpful in identifying infectious agents.
- It helps in restricting fragment length polymorphism.
- It has the ability to identify a single gene in a pool of DNA fragments.
- It helps detect cancer and genetic diseases like sickle cell mutation and monoclonal leukemia. (1, 3, 5, and 8)
How is Southern blotting used in forensics?
Forensic laboratories used southern blotting method to detect the smallest quantity of DNA, especially in the case of rape, thieves, or identification of parenthood. (7, 8)
Dna Slot Blot Protocol Igg
Image 6: Southern blotting is a procedure used in a forensic setting such as in the case of rape and other types of crimes that may require identification of DNA samples.
Picture Source: newyorker.com
Southern blot is a method commonly used in molecular biology. It has been a widely used technique for over three decades. Through southern blot, researchers can thoroughly understand the fundamentals of molecular biology. The primary purpose of southern blot is to detect a sequence of DNA in a given DNA sample.
Dna Slot Blot Protocol Definition
However, the southern blotting method can be quite expensive and extremely technical in nature. Sample-wise, it needs a large quantity of DNA, which is not always favorable in some settings.
With the advancement in the field of science and medicine, new DNA testing methods were developed such as PCR. It is better than southern blotting because the procedure involved is very straightforward. It is easy, fast, reliable, and only requires a small volume of DNA. (2, 4, 6, and 9)
Dna Slot Blot Protocol Assay