Dot Blot

Definition:
. A method for detecting a specific sequence of nucleotides in a DNA or RNA molecule. The nucleic acid sample is adsorbed onto a nitrocellulose filter and an appropriate gene probe, which matches the specific sequence being investigated, is added. After a period of incubation any excess probe is washed off and the nucleotide sequence under investigation can be detected by autoradiography. The technique offers significant savings in time, as chromatography or gel electrophoresis, and the complex blotting procedures for the gel are not required. However, it offers no information on the size of the target biomolecule. Furthermore, if two molecules of different sizes are detected, they will still appear as a single dot. Dot blots therefore can only confirm the presence or absence of a biomolecule or biomolecules which can be detected by the DNA probes or the antibody. A rapidquick and dirtyhybridization technique for semi quantifying a specific RNA and DNA fragment in a specimen without performing a more time-consuming Northern and Southern blot

Schematic of the use of two ASO probes on duplicate Dot-blot filters.

ASO:
ALLELE-SPECIFIC OLIGONUCLEOTIDE (ASO) is a short piece of synthetic DNA complementary to the sequence of a variable target DNA. It acts as a probe for the presence of the target in a Southern blot assay or, more commonly, in the simpler Dot blot assay. It is a common tool used in genetic testing, forensics, and Molecular Biology research.

Dot blot protocol:

Materials:
-Primary and Hrp conjugated secondary antibodies - Nitocellulose membrane - ECL solution - Plastic film wrap and x-ray film

Buffers:
- TBS: 20 mM Tris-hcl, 150mM NaCl, pH 7.5 - TBS-T: 0.05% Tween20 in TBS - Blocking solution : 5% BSA in TBS-T

Procedure:
- Label nitro cellulose membrane, spot slowly 2ul of sample on the membrane and allow it to dry. - Incubate the membrane in blocking solution for 1 hr at room temperature. - Incubate with primary antibody for 30 mins at RT. - Wash the membrane 3 times with TBS-T. - Incubate the membrane with the HRP-conjugated secondary antibody for 30 mins at RT. - Wash the membrane 3 times with TBS-T, then once with TBS for 5 mins. - Incubate the membrane with the ECL solution for 1 min. Then apply the plastic film wrap and expose to x-ray film at different exposure times. - Lastly, compare the samples with standard to estimate the concentration.

Applications for Dot blot:

Genetic testing:
Used to test for genetic disorders which involve direct examination of the DNA molecule. Allows the genetic diagnosis of vulnerabilities to inherited diseases, and can also be used to determine a child's paternity (genetic father) or a person's ancestry. The human genomeis believed to contain around 20,000 - 25,000 genes. And it identifies changes in chromosomes, genes, or proteins. Most of the time, testing is used to find changes that are associated with inherited disorders. The results of a genetic test can confirm or rule out a suspected genetic condition or help determine a person's chance of developing or passing on a genetic disorder. Several hundred genetic tests are currently in use, and more are being developed.

Molecular biology research:

Is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry. Molecular biology chiefly concerns itself with understanding the interactions between the various systems of a cell, including the interactions between the different types of DNA, RNA and protein biosynthesis as well as learning how these interactions are regulated.

Forensics:
Takes advantage of the uniqueness of an individual's DNA to answer forensic questions such as paternity/maternity testing and placing a suspect at a crime scene

Southern Blot
Definition:
Is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples from different sources, and to identify the size of the restriction fragment that contains the sequence. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane (nitrocellulose or nylon membrane) and subsequent fragment detection by probe hybridization or separated fragments, used to produce an autoradiogram. (Is an image on an x-ray film or nuclear emulsion produced by the pattern of decay emissions (e.g., beta particles or gamma rays) from a distribution of a radioactive substance.)

Sir Edwin Southern

The genetic researcher developed a system in the mid 1970s to identify DNA, and his technique became a predecessor of DNA fingerprinting. The procedure became known as the Southern Blot.

Southern blotting protocol:

Materials:
DNA gel electrophoresis product 3mm Whatman filter paper Nylon membrane Plastic wrap Paper towels Glass plate weights

Probe X-ray film Equipments: Hybridization oven, Rotator/ shaker, UV transilluminator, Incubator

Reagents:
Denaturing solution: - NaOH 0.5N - NaCl 1.5M Neutralizing solution: Tris 1M; NaCl 3M; pH 7.0 Wash buffer: SSC 2X; SDS .01% Transfer buffer: - 3M NaCl 175.3g - 0.15 Na Citrate - dH20 88.2 g

800 mls

- pH 7.0 with HCl (0.1 - 0.5 mLs approx) - 10x SSC Prehybridization buffer: - SSC - Denhardts 5x - SDS 0.5% 6x

- Denatured DNA 100ug/mL - Formide 50% Hybridization buffer: - SSC 6x - SDS 0.5% - Denatured DNA 100ug/mL - Formide 50% Probe: - Isotope labeled dCTP

Procedure:
- Load DNA samples (restriction enzyme treated) in 0.8% TBE gel Rock the gel in denaturing solution for 30 mins. (denaturing solution: NaOH 0.5N; NaCl 1.5M) Remove the buffer and rock the gel in neutralizing solution for 30 mins. (neutralizing solution: Tris 1M; NaCl 3M; pH 7.0)

Southern transfer: Set up transfer Place Whatman 3MM Filter paper Soak filter paper in transfer buffer (10X SSC) Place 2 sheets of wet filter paper Remove any air bubbles Place the gel Put a nylon on the membrane Flood the membrane with transfer buffer Cover with 2 sheets of wet whatman 3MM Filter paper Remove any air bubbles Cover with plastic wrap Stack paper towels above the gel Cover with a glass plate and add a weight Allow to transfer to go over night Remove paper towels and filter paper Rinse nylon membrane in 6x SSC Place membrane on a UV transilluminator to cross-link

Hybridization: Prehybridize membrane in prehybridization buffer Incubate at 42c for 2-4 hours Add hybridization buffer Add probe (isotope-labeled dCTP) Hybridize overnight Remove the solution Wash the membrane by wash buffer (2x SSC , 0.1% SDS-sodium dodecyl sulfate) Incubate at 52c for 30 mins (repeat wash step for 3 times) Expose to film. Incubate cassette at 37C for 10 minutes, then return to RT. Exposures vary from blot to blot (1' to 2 hr).

Applications:
DNA Fingerprinting:
(Also called DNA testing, DNA typing, or genetic fingerprinting) is a way of identifying a specific individual, rather than simply identifying a species or some particular trait, is currently used both for identifying paternity or maternity and for identifying criminals or victims. There is discussion of using DNA fingerprinting as a sort of personal identifier as well, although the viability of this is debatable. Although 99.9% of human DNA sequences are the same in every person, enough of the DNA is different to distinguish one individual from another, unless they are monozygotic twins. DNA profiling uses repetitive ("repeat") sequences that are highly variable called variable number tandem repeats (VNTRs), particularly short tandem repeats (STRs). VNTR loci are very similar between closely related humans, but so variable that unrelated individuals are extremely unlikely to have the same VNTRs.

Gene Expression:
The process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA (rRNA), transfer RNA (tRNA) or small nuclear RNA (snRNA) genes, the product is a functional RNA.

Dot Blot or Slot Blot:

A method for detecting a specific sequence of nucleotides in a DNA or RNA molecule.

Microarray analysis:
(Also commonly known as gene chip, DNA chip, or biochip) A tool used to sift through and analyze the information contained within a genome. A microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead.

Forensics:
Takes advantage of the uniqueness of an individual's DNA to answer forensic questions such as paternity/maternity testing and placing a suspect at a crime scene.

Other blotting methods:

Northern blot:
Is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample, are not used very often for diagnostic purposes; they are used mainly in research. The techniques are fairly sophisticated and other methods yield acceptable results (such as Southern blots or PCR). However, a description of Northern blots is included here in the interest of completeness and the probability that you will run across the term in some of the papers you may read.

Western blot:
Is a widely used analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract.

Far western blot:

Is a molecular biological method which is based on the technique of Western blotting. While usual Western blotting uses an antibody to detect a protein of interest, far-Western blotting uses a non-antibody protein, which can bind the protein of interest. Thus, whereas Western blotting is used for the detection of certain proteins, far-Western blotting is rather employed to detect protein:protein interactions .

Eastern blot:
Is a biochemical technique used to analyze protein post translational modifications (PTM) such as lipids and glycoconjugates. It is most often used to detect carbohydrate epitopes. Thus, Eastern blotting can be considered an extension of the biochemical technique of Western blotting.

Far eastern blot:

Used for the analysis of lipids separated by high-performance thin layer chromatography (HPTLC). It was established as a method for transferring lipids from an HPTLC plate to a polyvinyledene difluoride (PVDF) membrane within a minute. Applications of this with other methods have been studied. Far-eastern blotting allows for the following techniques:

Purification of glycosphingolipids and phospholipids. Structural analysis of lipids in conjunction with direct mass spectrometry. Binding study using various ligands such as antibodies, lectins, bacterium, viruses, and toxins, and Enzyme reaction on membranes.

South western blot:

Is a lab technique which involves identifying and characterizing DNA-binding proteins (proteins that bind to DNA) by their ability to bind to specific oligonucleotide probes, The proteins are separated by gel electrophoresis and are subsequently transferred to nitrocellulose membranes similar to other types of blotting. The name southwestern blotting is based on the fact that this technique detects DNA-binding proteins, since DNA detection is by Southern blotting and protein detection is by western blotting.

References:
http://en.wikipedia.org/wiki/Dot_blot http://www.encyclopedia.com/doc/1O6-dotblot.html http://medical-dictionary.thefreedictionary.com/Dot+Blot http://en.wikipedia.org/wiki/Allele-specific_oligonucleotide http://www.abcam.com/ps/pdf/protocols/Dot%20blot%20protocol.pdf http://www.youtube.com/watch?v=EzEeWMOZr3c http://en.wikipedia.org/wiki/Genetic_testing http://www.lbl.gov/Education/ELSI/genetic-testing.html http://en.wikipedia.org/wiki/Molecular_biology http://www.ornl.gov/sci/techresources/Human_Genome/elsi/forensics.shtml http://en.wikipedia.org/wiki/Southern_blot http://medical-dictionary.thefreedictionary.com/Southern+Blot

http://en.wikipedia.org/wiki/Edwin_Southern http://scienceheroes.com/index.php?option=com_content&view=article&id=523&Itemid=538 http://www.mun.ca/biology/scarr/Gr12-18.html http://www.med.upenn.edu/mcrc/parmacek_lab/SouthernBlot-singlecopy.shtml http://labs.fhcrc.org/gottschling/General%20Protocols/southerns.html http://www.youtube.com/watch?v=3I9wzwj0b_A http://www.wisegeek.com/what-is-dna-fingerprinting.htm http://en.wikipedia.org/wiki/DNA_profiling http://en.wikipedia.org/wiki/Gene_expression http://www.news-medical.net/health/What-is-Gene-Expression.aspx http://www.medterms.com/script/main/art.asp?articlekey=30712 http://en.wikipedia.org/wiki/DNA_microarray

http://en.wikipedia.org/wiki/Forensic_science http://en.wikipedia.org/wiki/Northern_blot http://www.escience.ws/b572/L13/north.html http://en.wikipedia.org/wiki/Western_blot http://en.wikipedia.org/wiki/Far-western_blotting http://en.wikipedia.org/wiki/Eastern_blotting http://en.wikipedia.org/wiki/Far-Eastern_blotting http://en.wikipedia.org/wiki/Southwestern_blot