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Gel electrophoresis

Because nucleic acids are negatively charged ions at neutral or alkaline pH in an aqueous environment, they can be moved by an electric field. Gel electrophoresis is a technique used to separate charged molecules on the basis of size and charge. The nucleic acids can be separated as whole chromosomes or as fragments. The nucleic acids are loaded into a slot at one end of a gel matrix, an electric current is applied, and negatively charged molecules are pulled toward the opposite end of the gel (the end with the positive electrode). Smaller molecules move through the pores in the gel faster than larger molecules; this difference in the rate of migration separates the fragments on the basis of size. The nucleic acids in a gel matrix are invisible until they are stained with a compound that allows them to be seen, such as a dye. Distinct fragments of nucleic acids appear as bands at specific distances from the top of the gel (the negative electrode end) that are based on their size ( [link] ). A mixture of many fragments of varying sizes appear as a long smear, whereas uncut genomic DNA is usually too large to run through the gel and forms a single large band at the top of the gel.

Photo shows a black background with 9 faint gray vertical bands (lanes). In those bands are horizontal white slightly blurry bands of varying thicknesses and brightness. The faint gray lanes on the left and right edges have a lot of horizontal bands, and the 7 in the middle have only a few each, in different positions.
Shown are DNA fragments from six samples run on a gel, stained with a fluorescent dye and viewed under UV light. (credit: modification of work by James Jacob, Tompkins Cortland Community College)

Polymerase chain reaction

DNA analysis often requires focusing on one or more specific regions of the genome. It also frequently involves situations in which only one or a few copies of a DNA molecule are available for further analysis. These amounts are insufficient for most procedures, such as gel electrophoresis. Polymerase chain reaction (PCR) is a technique used to rapidly increase the number of copies of specific regions of DNA for further analyses ( [link] ). PCR uses a special form of DNA polymerase, the enzyme that replicates DNA, and other short nucleotide sequences called primers that base pair to a specific portion of the DNA being replicated. PCR is used for many purposes in laboratories. These include: 1) the identification of the owner of a DNA sample left at a crime scene; 2) paternity analysis; 3) the comparison of small amounts of ancient DNA with modern organisms; and 4) determining the sequence of nucleotides in a specific region.

Figure showing PCR in 4 steps. First, the double strand of DNA is denatured at 95 degrees Celsius to separate the strands. The 2 strands are then annealed at approximately 50 degrees Celsius using primers. DNA polymerase then extends the new strands at 72 degrees Celsius. The fourth step shows that this procedure takes place many times, resulting in an increase in copies of the original DNA.
Polymerase chain reaction, or PCR, is used to produce many copies of a specific sequence of DNA using a special form of DNA polymerase.

Cloning

In general, cloning    means the creation of a perfect replica. Typically, the word is used to describe the creation of a genetically identical copy. In biology, the re-creation of a whole organism is referred to as “reproductive cloning.” Long before attempts were made to clone an entire organism, researchers learned how to copy short stretches of DNA—a process that is referred to as molecular cloning.

Molecular cloning

Cloning allows for the creation of multiple copies of genes, expression of genes, and study of specific genes. To get the DNA fragment into a bacterial cell in a form that will be copied or expressed, the fragment is first inserted into a plasmid. A plasmid    (also called a vector in this context) is a small circular DNA molecule that replicates independently of the chromosomal DNA in bacteria. In cloning, the plasmid molecules can be used to provide a "vehicle" in which to insert a desired DNA fragment. Modified plasmids are usually reintroduced into a bacterial host for replication. As the bacteria divide, they copy their own DNA (including the plasmids). The inserted DNA fragment is copied along with the rest of the bacterial DNA. In a bacterial cell, the fragment of DNA from the human genome (or another organism that is being studied) is referred to as foreign DNA to differentiate it from the DNA of the bacterium (the host DNA).

Questions & Answers

Why does water move through a membrane?
Christina Reply
How many bones are in the human skeleton
Treasure Reply
203
Oyeleke
procce of digestion of proteins a long human alimentarycanal
Carson Reply
what are the properties of lipids?
Isiah Reply
They are: Fatty acids, fats, oils, waxes, phospholipid, glycolipids, steroids and some vitamins
Rachel
explain why a fresh water fish excrete ammonia
Leonard Reply
plz answer my question
Leonard
sorry i meant it has a nucleous unlike plant cells lol
Lailah
Ammonia is the end product of protein catabolism and is stored in the body of the fish in high concentrations relative to basal excretion rates. Ammonia, if allowed to accumulate, is toxic and is converted to less toxic compounds or excreted
Rachel
What are eukaryotic cells?
Nwosueke Reply
cell with no nucleous so not a plant cell
Lailah
eukaryotic cells are membrane bound organelles that have a membrane bound nucleus
ojeen
where does the cell get energy for active transport processes?
A'Kaysion Reply
IDK maybe glucose
Lailah
what is synapsis
Adepoju Reply
how many turns are required to make a molecule of sucrose in Calvin cycle
Amina Reply
why Calvin cycle occurs in stroma
Amina
why do humans enhale oxygen and exhale carbondioxide?
Maryam Reply
why do humans enhale oxygen and exhale carbondioxide? For the purpose of breaking down the food
dil
what is allele
uzoka Reply
process of protein synthesis
SANTOSH Reply
what is cell
Zulf Reply
a cell is a smallest basic, structural and functional unit of life that is capable of self replication
Lucas
why does a fresh water fish excrete ammonia
Leonard
plz answer my question
Leonard
Ammonia is a toxic colorless gas and when its inside the fish biological system is converted to a less toxic compound then excreted in the form of urea. However too much ammonia will kill the fish " Ammonia Poisoning " which is a very common disease among fish.
This
what is cytoplasm
uzoka Reply
cytoplasm is fluid of cell.
Deepak
how many major types of Cloning
Saeed Reply
two
amir
two
Zulf

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Source:  OpenStax, Concepts of biology. OpenStax CNX. Feb 29, 2016 Download for free at http://cnx.org/content/col11487/1.9
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