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This illustration shows the four main steps of DNA extraction. In the first step, cells in a test tube are lysed using a detergent that disrupts the plasma membrane. In the second step, cell contents are treated with protease to destroy protein, and RNAase to destroy RNA. The resulting slurry is centrifuged to pellet the cell debris. The supernatant, or liquid, containing the DNA is then transferred to a clean test tube. The DNA is precipitated with ethanol. It forms viscous, mucous-like strands that can be spooled on a glass rod
This diagram shows the basic method used for extraction of DNA.

RNA analysis is performed to study gene expression patterns in cells. RNA is naturally very unstable because RNAses are commonly present in nature and very difficult to inactivate. Similar to DNA, RNA extraction involves the use of various buffers and enzymes to inactivate macromolecules and preserve the RNA.

Gel electrophoresis

Because nucleic acids are negatively charged ions at neutral or basic pH in an aqueous environment, they can be mobilized by an electric field. Gel electrophoresis is a technique used to separate molecules on the basis of size, using this charge. The nucleic acids can be separated as whole chromosomes or fragments. The nucleic acids are loaded into a slot near the negative electrode of a semisolid, porous gel matrix and pulled toward the positive electrode at the opposite end of the gel. 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. There are molecular weight standard samples that can be run alongside the molecules to provide a size comparison. Nucleic acids in a gel matrix can be observed using various fluorescent or colored dyes. Distinct nucleic acid fragments appear as bands at specific distances from the top of the gel (the negative electrode end) on the basis of their size ( [link] ). A mixture of genomic DNA 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 an agarose gel illuminated under UV light. The gel contains nine lanes from left to right. Each lane was loaded with a sample containing DNA fragments of differing size that separated as they travelled through the gel from top to bottom. The DNA appears as thin, white bands on a black background. Lanes one and nine contain many bands from a DNA standard. These bands are closely spaced toward the top, and spaced farther apart further down the gel. Lanes two through eight contain one or two bands each. Some of these bands are identical in size and run the same distance into the gel. Others run a slightly different distance, indicating a small difference in size.
Shown are DNA fragments from seven samples run on a gel, stained with a fluorescent dye, and viewed under UV light. (credit: James Jacob, Tompkins Cortland Community College)

Amplification of nucleic acid fragments by polymerase chain reaction

Although genomic DNA is visible to the naked eye when it is extracted in bulk, DNA analysis often requires focusing on one or more specific regions of the genome. Polymerase chain reaction ( PCR ) is a technique used to amplify specific regions of DNA for further analysis ( [link] ). PCR is used for many purposes in laboratories, such as the cloning of gene fragments to analyze genetic diseases, identification of contaminant foreign DNA in a sample, and the amplification of DNA for sequencing. More practical applications include the determination of paternity and detection of genetic diseases.

Illustration shows the amplification of a DNA sequence by the polymerase chain reaction. PCR consists of three steps—denaturation, annealing, and DNA synthesis—that occur at high, low, and intermediate temperatures. In step 1, the denaturation step, the sample is heated to a high temperature so the DNA strands separate. In step 2, annealing, the sample is cooled so two primers can anneal to the two strands of DNA. The primers are spaced such that the sequence of interest between them will be amplified. In step 3, DNA synthesis, the sample is warmed to the optimal temperature for Taq polymerase, which synthesizes the complementary strand from the primer to the 3' end of the molecule. This cycle is repeated again and again. Each time, the newly synthesized strands serve as templates so that the amount of DNA doubles with each cycle. As the cycles continue, more and more strands are the size of the distance between the two primers; in the end, the vast majority of strands are this size.
Polymerase chain reaction, or PCR, is used to amplify a specific sequence of DNA. Primers—short pieces of DNA complementary to each end of the target sequence—are combined with genomic DNA, Taq polymerase, and deoxynucleotides. Taq polymerase is a DNA polymerase isolated from the thermostable bacterium Thermus aquaticus that is able to withstand the high temperatures used in PCR. Thermus aquaticus grows in the Lower Geyser Basin of Yellowstone National Park. Reverse transcriptase PCR (RT-PCR) is similar to PCR, but cDNA is made from an RNA template before PCR begins.

Questions & Answers

what is fertilization?
Muhamed Reply
What kind of nutrients is composed of plants
Annie Reply
phosphorus,potassium,nitrogen
Kritika
what is hormones
Igwe Reply
hormon is the chemical messanger
Sneha
Genes can make someone dull?
Taperah
21ecological instrument and their diagrams
Ayomide Reply
what are the kidney disease
Immaculate Reply
kidney stones
Gracelyne
it is a disease that affects the kidney
Miriam
what are some lung diseases
Gracelyne
what is micro-organism
Jackson Reply
what is the hypothesis
Jackson
hypothesis is a proposed explanation for a phenomenon
Miriam
hypothesis is raw materials
KP
what is biology
KP
biology is the study of living things and their interaction with their environment
Miriam
what does mean stigma
Amira Reply
what is the full of the MOST dangerous disease in the world where one stops sleeping and just dies :Hint ; FFI
God Reply
fatal familial insomnia which affects the thalamus
Miriam
there are other dangerous diseases like CAD i.e coronary artery disease
Miriam
what is matter
Thomas Reply
it is any thing that has weight and occupies space
Anye
matter is any substances that occupies spaces and has mass
Jackson
describe photosynthesis
Mavis Reply
What is equilibrium
Mavis
What is equilibrium
Mavis
like corporal intern balance right?
FRANCISCA
on my own understanding is just a balanced state
Stanley
photosynthesis is the process by which plants and other organisms convert light energy to chemical energy
Miriam
what is a chromosome?
Wise Reply
Are thread-like structures located inside the nucleus of animal and plant cells.
Canab
thx
Wise
what are the difference between Biotic community and Ecological nitche.
Ganiyat Reply
what is the celll
KAMOLIKA Reply
A cell is the simplest bit of living matter that exist independently
Ganiyat
cell is the basic unit of life
Shadrack
what is ecdysis
Shadrack
what is genetics
Sebastian Reply
The cell is the simplest bit of living matter that can exist independently.
Ganiyat
what happenes when the cell of an organism Is removed?
Isaac Reply
The cell will not function properly
Eunice

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Source:  OpenStax, Biology. OpenStax CNX. Feb 29, 2016 Download for free at http://cnx.org/content/col11448/1.10
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