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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 metabolism
Mlungisi Reply
pls what is the chemical symbol for Methane
Afia Reply
CH4
Francis
ch4
Paolo
what is life?
Josephus
What do you think is life?
Isala
Hi
Isaac
hey
Isala
what is DNA
Machuol
all living things have certain characteristics in common which are referred to as dash
IBUKUN Reply
classifying living things in the world in two major groups
IBUKUN
mention the seven characteristics that distinguish living things from nonliving things
IBUKUN
which of the characteristics of living things involved taking in the use of seed by animals as well as the taking in of mineral sentences and their uses by plants
IBUKUN
Life process
Afia
Growth, Respiration, Excretion, Movement, Sensitivity, Nutrition,and Reproductive
Afia
Nutrition
Afia
nutrition
Lawrence
Mendel experiment when years ago in work
Biruk Reply
what is the smallest unit in an organism
Neimar Reply
what is endoplasmic
Fatou Reply
If Jane was in room(B) while her mother Stella was in room (Y). Jane was cooking fish, her mother came to smell the good scent, By what process did her mother came to smell the scent
Neimar Reply
Diffusion
Afia
Is it a serious question?
Ehsan Reply
what's the question
Joseph
how many days do a bean seed take to germinate
Nando
no idea
Afia
what is DNA
Yahaya Reply
genetic information
MG
Deoxyribonucliec acid (DNA) is the cell's hereditary material that contains instructions for growth, development and reproduction.
Joseph
ok
oly
what's different between sex and gender
oly
Are there differences between sex and gender?
Theo
lol
Andrew
hi Yahaya its Deoxyribonucleic acid
Neimar
describe an experiment to show that plants require light for photosynthesis
Diyara Reply
uuh... putting a plant in a dark closet... and another in a light enviroment?
Anastasiya
wow! awesome explanation Anastasiya.
Joseph
please can you explain why air is homogenous
Joseph
Because each layer of the Earth's atmosphere has a different density, each layer of air is it's own homogenous.
MG
tell me about big bang
Mustafa
what's DNA
Mustafa
What's an amoeba
Bigger Reply
An amoeba is a cell or an organism that has the ability to ulter it's shape.
Joseph
An amoeba has an irregular shape and it changes constantly,it's a unicellular organism belong to a group called protists it has a pseudopodia used for locomotion and ingestion...
Emmanuel
amoeba is an organism that has an inregular shape which changes constantly
Cashizz
amoeba is an unicellular organism that uses pseudopodia,it does not have a constant shape
Alohan
what is the difference between DNA and RNA
Alohan
DNA with oxygenated but RNA without oxygenated
qax
what is the mode of nutrition of fungi
Survive
asexual
qax
amoeba are protozoa with one cell and no fixed shape
James
heterotrophic and outrotrophic
qax
what is the between arteries and capillary
qax
arteries>arterioles>capillaries. decrease in size and thus pressure
Anastasiya
DNA deossoribonucleic acid. RNA ribonucleic acid. The difference between the two lies in a lack of one oxygen on the sugar in DNA. Also: in DNA the azotate bases are Guanine, Citosine, Adenine and TIMINE base, the latter is replaced by URACILE in RNA. DNA formes a double helix structure...
Anastasiya
... while RNA form is usually a single stand line, but it can form loops etc
Anastasiya
what is a zygote?
Darius Reply
zygote is a eukaryotic cell formed by a fertilization event between two gametes. The zygote's genome is a combination of the DNA in each gamete, and contains all of the genetic information necessary to form a new individual.
MG
A zygote is an organism formed after fertilization
Neimar
how do plants form a zygote
Paclina Reply
What is zygote
Van
what is zygote
Darius
Zygote is located inside the ovule, which is present in the ovary.
MG
zygote is a eukaryotic cell formed by a fertilization event between two gametes. The zygote's genome is a combination of the DNA in each gamete, and contains all of the genetic information necessary to form a new individual.
MG
Okay
Van
what is biology?
Aadan Reply
biology is the study of living n non living organism
Kelsia
what is procotist?
Kelsia
Biology is the branch of science that deals with the study of living and non living things
Neimar
Describe at least two major changes to the animal phylogenetic tree that have come about due to molecular or genetic findings.
Tamala Reply

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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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