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Due to its negatively charged backbone, DNA is strongly attracted to a positive electrode. In agarose gel electrophoresis, the gel is oriented horizontally in a buffer solution. Samples are loaded into sample wells on the side of the gel closest to the negative electrode, then drawn through the molecular sieve of the agarose matrix toward the positive electrode. The agarose matrix impedes the movement of larger molecules through the gel, whereas smaller molecules pass through more readily. Thus, the distance of migration is inversely correlated to the size of the DNA fragment, with smaller fragments traveling a longer distance through the gel. Sizes of DNA fragments within a sample can be estimated by comparison to fragments of known size in a DNA ladder also run on the same gel. To separate very large DNA fragments, such as chromosomes or viral genomes, agarose gel electrophoresis can be modified by periodically alternating the orientation of the electric field during pulsed-field gel electrophoresis (PFGE) . In PFGE , smaller fragments can reorient themselves and migrate slightly faster than larger fragments and this technique can thus serve to separate very large fragments that would otherwise travel together during standard agarose gel electrophoresis. In any of these electrophoresis techniques, the locations of the DNA or RNA fragments in the gel can be detected by various methods. One common method is adding ethidium bromide , a stain that inserts into the nucleic acids at non-specific locations and can be visualized when exposed to ultraviolet light. Other stains that are safer than ethidium bromide, a potential carcinogen, are now available.

a) A diagram of the process of agarose gel electrophoresis. 1 – An agarose and buffer solution is heated and poured into a form. This result in a rectangular block with indents along one end labeled “S=sample wells”. 2 – When cooled, the agarose gel block contains small wells (S) where the sample will be place. 3 – Each sample is added to a separate well. Then the agarose gel is placed in a chamber that generates a charge across the gel. The samples are added using micropipettes. 4 – The solution within the chamber conducts the electric current generated by the chamber. The side nearest the sample well will have a negative charge; the other side will have a positive charge. 5 – DNA has a negative charge and will be drawn to the positive pole of the gel. Smaller DNA molecules will be able to travel faster through the matrix of the gel. 6 – One well will contain a DNA ladder, which has fragments of known size. This ladder is used to identify the sizes of the bands in the sample. The ladder looks like many bands in the gel; from top to bottom the sizes of the bands are – 2000 bp, 15000 bp, 1000 bp, 750 bp, 500 bp, 250 bp. The other lanes have a few bands of various sizes.
(a) The process of agarose gel electrophoresis. (b) A researcher loading samples into a gel. (c) This photograph shows a completed electrophoresis run on an agarose gel. The DNA ladder is located in lanes 1 and 9. Seven samples are located in lanes 2 through 8. The gel was stained with ethidium bromide and photographed under ultraviolet light. (credit a: modification of work by Magnus Manske; credit b: modification of work by U.S. Department of Agriculture; credit c: modification of work by James Jacob)

Restriction fragment length polymorphism (rflp) analysis

Restriction enzyme recognition sites are short (only a few nucleotides long), sequence-specific palindromes, and may be found throughout the genome. Thus, differences in DNA sequences in the genomes of individuals will lead to differences in distribution of restriction-enzyme recognition sites that can be visualized as distinct banding patterns on a gel after agarose gel electrophoresis. Restriction fragment length polymorphism (RFLP) analysis compares DNA banding patterns of different DNA samples after restriction digestion ( [link] ).

RFLP analysis has many practical applications in both medicine and forensic science . For example, epidemiologists use RFLP analysis to track and identify the source of specific microorganisms implicated in outbreaks of food poisoning or certain infectious diseases. RFLP analysis can also be used on human DNA to determine inheritance patterns of chromosomes with variant genes, including those associated with heritable diseases or to establish paternity .

Questions & Answers

what is mutation
Cynthia Reply
alteration in genetic makeup
UDEME
Hi, I'm new here. I'm Bello Abdul Hakeem from Nigeria.
BELLO Reply
welcome on board
Kamaluddeen
Thanks brother I'm an undergraduate. I hope to study for MBBS.
BELLO
pls guys help me out
Linda
what is limitation of plate
Linda
limitation of plate load test . there are some that should be considered while performing load test which are given below, this test is usually performed on relatively similar plate ,usually 1 or 2 square foot area the reason is that the plate of greater are the economically not feasible
yemi
Mechanism of bacterial pathogenicity
rashida Reply
Toxicity
Ibrahim
write a short note on Algea
najaatu Reply
green minutes plants organisms that are produced in turf.
Prince
Algae is a kind of a photosynthetic organism, which is usually grown in the moist areas. These are usually the simple plants that grow near to the water bodies. It contains a kind of chlorophyll pigments that act as a primary coloring agent.
Avi
they are eukaryotic and most lived in fresh water. they are photosynthetic that's, they contain chlorophyll and store starch in discrete chloroplast.
Prince
are bacteria important to man
just Reply
yes, depending on the type of bacteria .eg the normal florals, the latic acid batteries etc are important to man
Lois
I mean latic acid bacterial
Lois
I don't understand this topic
Jane Reply
what topic is that
Rose
sahi kon hai bhosri ke
saurabh Reply
define metabolism of carbohydrates with example
Thavasi Reply
what is sterilization a
Sani
the process of keep equipment free from bacteria
shar
is it only bacteria?
Lois
no undesirable fungi and contamination also.
Khushbu
what is streak plate method
Offikwu
what is the biofilm
Dimingu
metabolism is the sum of all the biochemical reaction required for energy generation and use of that energy to synthesize cell materials from small molecules in environment.
Alex
boclenia
Pooja
what are granulocytes
Shawnitta Reply
e.coli
Sukhdeep
granulocytes are type of WBCs which contains granules in the cytoplasm
Owili
what is mutation
Cynthia
it is the interchange of genes from their normal sequence
Esther
is an heritable change of the base-pair sequence of genetic material
Elizabeth
suitable example for prokaryotes
Suvetha Reply
one of the possible early sources of energy was
Suvetha
uv radiation and lighting
Anisha
e coli is the example of prokaryotes
Sukhdeep
archaea too
Noel
which is the specific virus causing typhoid
Jeremiah Reply
it's caused by a virulent bacteria called Salmonella Typhi
Sarah
write the life cycle of HIV
Firomsa Reply
describe the internal and external structure of prokaryotic cell in terms of there appearance and functions
Lenia Reply
compare and contrast similar structures found in prokaryotic and eukaryotic cells
Lenia
control of microorganisms
ISTEFAZUL Reply
what is sterilization
Sani
how can a doctor treat a person affected by endospore forming bacteria in his/her wound?
Mambo Reply
define disinfectant
Sani
the process of killing
shar
the process of killing microorganisms
shar

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Source:  OpenStax, Microbiology. OpenStax CNX. Nov 01, 2016 Download for free at http://cnx.org/content/col12087/1.4
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