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DNA fragments can also be amplified from an RNA template in a process called reverse transcriptase PCR (RT-PCR)    . The first step is to recreate the original DNA template strand (called cDNA) by applying DNA nucleotides to the mRNA. This process is called reverse transcription. This requires the presence of an enzyme called reverse transcriptase. After the cDNA is made, regular PCR can be used to amplify it.

Deepen your understanding of the polymerase chain reaction by clicking through this interactive exercise .

Hybridization, southern blotting, and northern blotting

Nucleic acid samples, such as fragmented genomic DNA and RNA extracts, can be probed for the presence of certain sequences. Short DNA fragments called probes are designed and labeled with radioactive or fluorescent dyes to aid detection. Gel electrophoresis separates the nucleic acid fragments according to their size. The fragments in the gel are then transferred onto a nylon membrane in a procedure called blotting ( [link] ). The nucleic acid fragments that are bound to the surface of the membrane can then be probed with specific radioactively or fluorescently labeled probe sequences. When DNA is transferred to a nylon membrane, the technique is called Southern blotting    , and when RNA is transferred to a nylon membrane, it is called northern blotting    . Southern blots are used to detect the presence of certain DNA sequences in a given genome, and northern blots are used to detect gene expression.

In Southern blotting, DNA is separated on the basis of size by agarose gel electrophoresis. The fragments run through the gel from top to bottom. In the gel shown in this figure, there are so many DNA fragments they appear as a smear in each lane.  The DNA from the gel is transferred to a nylon membrane. To do so, the gel is sandwiched between filter paper and the membrane and placed in hybridization buffer. Paper towels above the gel wick up the moisture and assist in the transfer. The nylon membrane is then incubated with a radioactively or fluorescently labeled probe that is complementary to the sequence of interest. Discrete bands appear where the sequence of interest is located.
Southern blotting is used to find a particular sequence in a sample of DNA. DNA fragments are separated on a gel, transferred to a nylon membrane, and incubated with a DNA probe complementary to the sequence of interest. Northern blotting is similar to Southern blotting, but RNA is run on the gel instead of DNA. In western blotting, proteins are run on a gel and detected using antibodies.

Molecular cloning

In general, the word “cloning” means the creation of a perfect replica; however, 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 reproduce desired regions or fragments of the genome, a process that is referred to as molecular cloning.

Cloning small fragments of the genome allows for the manipulation and study of specific genes (and their protein products), or noncoding regions in isolation. A plasmid (also called a vector) is a small circular DNA molecule that replicates independently of the chromosomal DNA. In cloning, the plasmid molecules can be used to provide a "folder" in which to insert a desired DNA fragment. Plasmids are usually introduced into a bacterial host for proliferation. In the bacterial context, the fragment of DNA from the human genome (or the genome of another organism that is being studied) is referred to as foreign DNA    , or a transgene, to differentiate it from the DNA of the bacterium, which is called the host DNA    .

Plasmids occur naturally in bacterial populations (such as Escherichia coli ) and have genes that can contribute favorable traits to the organism, such as antibiotic resistance    (the ability to be unaffected by antibiotics). Plasmids have been repurposed and engineered as vectors for molecular cloning and the large-scale production of important reagents, such as insulin and human growth hormone. An important feature of plasmid vectors is the ease with which a foreign DNA fragment can be introduced via the multiple cloning site (MCS)    . The MCS is a short DNA sequence containing multiple sites that can be cut with different commonly available restriction endonucleases. Restriction endonucleases recognize specific DNA sequences and cut them in a predictable manner; they are naturally produced by bacteria as a defense mechanism against foreign DNA. Many restriction endonucleases make staggered cuts in the two strands of DNA, such that the cut ends have a 2- or 4-base single-stranded overhang. Because these overhangs are capable of annealing with complementary overhangs, these are called “sticky ends.” Addition of an enzyme called DNA ligase permanently joins the DNA fragments via phosphodiester bonds. In this way, any DNA fragment generated by restriction endonuclease cleavage can be spliced between the two ends of a plasmid DNA that has been cut with the same restriction endonuclease ( [link] ).

Questions & Answers

where we get a research paper on Nano chemistry....?
Maira Reply
what are the products of Nano chemistry?
Maira Reply
There are lots of products of nano chemistry... Like nano coatings.....carbon fiber.. And lots of others..
Even nanotechnology is pretty much all about chemistry... Its the chemistry on quantum or atomic level
no nanotechnology is also a part of physics and maths it requires angle formulas and some pressure regarding concepts
Preparation and Applications of Nanomaterial for Drug Delivery
Hafiz Reply
Application of nanotechnology in medicine
what is variations in raman spectra for nanomaterials
Jyoti Reply
I only see partial conversation and what's the question here!
Crow Reply
what about nanotechnology for water purification
RAW Reply
please someone correct me if I'm wrong but I think one can use nanoparticles, specially silver nanoparticles for water treatment.
yes that's correct
I think
Nasa has use it in the 60's, copper as water purification in the moon travel.
nanocopper obvius
what is the stm
Brian Reply
is there industrial application of fullrenes. What is the method to prepare fullrene on large scale.?
industrial application...? mmm I think on the medical side as drug carrier, but you should go deeper on your research, I may be wrong
How we are making nano material?
what is a peer
What is meant by 'nano scale'?
What is STMs full form?
scanning tunneling microscope
how nano science is used for hydrophobicity
Do u think that Graphene and Fullrene fiber can be used to make Air Plane body structure the lightest and strongest. Rafiq
what is differents between GO and RGO?
what is simplest way to understand the applications of nano robots used to detect the cancer affected cell of human body.? How this robot is carried to required site of body cell.? what will be the carrier material and how can be detected that correct delivery of drug is done Rafiq
analytical skills graphene is prepared to kill any type viruses .
Any one who tell me about Preparation and application of Nanomaterial for drug Delivery
what is Nano technology ?
Bob Reply
write examples of Nano molecule?
The nanotechnology is as new science, to scale nanometric
nanotechnology is the study, desing, synthesis, manipulation and application of materials and functional systems through control of matter at nanoscale
Is there any normative that regulates the use of silver nanoparticles?
Damian Reply
what king of growth are you checking .?
What fields keep nano created devices from performing or assimulating ? Magnetic fields ? Are do they assimilate ?
Stoney Reply
why we need to study biomolecules, molecular biology in nanotechnology?
Adin Reply
yes I'm doing my masters in nanotechnology, we are being studying all these domains as well..
what school?
biomolecules are e building blocks of every organics and inorganic materials.
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Source:  OpenStax, Genetics and evolution. OpenStax CNX. Aug 07, 2014 Download for free at https://legacy.cnx.org/content/col11595/1.2
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