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Recombinant DNA
From Wikipedia, the free encyclopedia

Construction of recombinant DNA, in which a foreign DNA fragment is inserted into a plasmid vector.
In this example, the gene indicated by the white color is inactivated upon insertion of the foreign
DNA fragment.

Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory

methods of genetic recombination (such as molecular cloning) to bring together genetic
material from multiple sources, creatingsequences that would not otherwise be found in
biological organisms. Recombinant DNA is possible because DNA molecules from all
organisms share the same chemical structure. They differ only in
the nucleotidesequence within that identical overall structure.
Contents
[hide]

1 Introduction

2 Creating recombinant DNA

3 Expression of recombinant DNA

4 Properties of organisms containing recombinant DNA

5 Applications of recombinant DNA technology

6 History of recombinant DNA

7 Controversy

8 See also

9 References
o

9.1 Further reading

10 External links

Introduction[edit]
Recombinant DNA is the general name for taking a piece of one DNA, combining it with
another strand of DNA. Recombinant DNA molecules are sometimes called chimeric
DNA, because they are usually made of material from two different species, like the
mythical chimera. R-DNA technology uses palindromic sequences and leads to the
production ofsticky and blunt ends.
The DNA sequences used in the construction of recombinant DNA molecules can
originate from any species. For example, plant DNA may be joined to bacterial DNA, or
human DNA may be joined with fungal DNA. In addition, DNA sequences that do not
occur anywhere in nature may be created by the chemical synthesis of DNA, and
incorporated into recombinant molecules. Using recombinant DNA technology and
synthetic DNA, literally any DNA sequence may be created and introduced into any of a
very wide range of living organisms.
Proteins that can result from the expression of recombinant DNA within living cells are
termed recombinant proteins. When recombinant DNA encoding a protein is introduced
into a host organism, the recombinant protein is not necessarily produced. [citation
needed]
Expression of foreign proteins requires the use of specialized expression vectors
and often necessitates significant restructure by foreign coding sequence. [citation needed]
Recombinant DNA differs from genetic recombination in that the former results from
artificial methods in the test tube, while the latter is a normal biological process that
results in the remixing of existing DNA sequences in essentially all organisms.

Creating recombinant DNA[edit]

Main article: Molecular cloning

Molecular cloning is the laboratory process used to create recombinant DNA. [1][2][3][4] It is
one of two widely used methods, along withpolymerase chain reaction (PCR) used to
direct the replication of any specific DNA sequence chosen by the experimentalist. The
fundamental difference between the two methods is that molecular cloning involves
replication of the DNA within a living cell, while PCR replicates DNA in the test tube, free
of living cells.
Formation of recombinant DNA requires a cloning vector, a DNA molecule that
replicates within a living cell. Vectors are generally derived from plasmids or viruses,
and represent relatively small segments of DNA that contain necessary genetic signals
for replication, as well as additional elements for convenience in inserting foreign DNA,

identifying cells that contain recombinant DNA, and, where appropriate, expressing the
foreign DNA. The choice of vector for molecular cloning depends on the choice of host
organism, the size of the DNA to be cloned, and whether and how the foreign DNA is to
be expressed.[5]The DNA segments can be combined by using a variety of methods,
such as restriction enzyme/ligase cloning or Gibson assembly.
In standard cloning protocols, the cloning of any DNA fragment essentially involves
seven steps: (1) Choice of host organism and cloning vector, (2) Preparation of vector
DNA, (3) Preparation of DNA to be cloned, (4) Creation of recombinant DNA, (5)
Introduction of recombinant DNA into the host organism, (6) Selection of organisms
containing recombinant DNA, and (7) Screening for clones with desired DNA inserts and
biological properties.[4] These steps are described in some detail in a related article
(molecular cloning).

Expression of recombinant DNA[edit]

Main article: Gene expression
Following transplantation into the host organism, the foreign DNA contained within the
recombinant DNA construct may or may not be expressed. That is, the DNA may simply
be replicated without expression, or it may be transcribed andtranslated so that a
recombinant protein is produced. Generally speaking, expression of a foreign gene
requires restructuring the gene to include sequences that are required for producing
an mRNA molecule that can be used by the host's translational
apparatus (e.g. promoter, translational initiation signal, and transcriptional terminator).
[6]
Specific changes to the host organism may be made to improve expression of the
ectopic gene. In addition, changes may be needed to the coding sequences as well, to
optimize translation, make the protein soluble, direct the recombinant protein to the
proper cellular or extracellular location, and stabilize the protein from degradation. [7]

Properties of organisms containing recombinant DNA [edit]

In most cases, organisms containing recombinant DNA have apparently
normal phenotypes. That is, their appearance, behavior and metabolism are usually
unchanged, and the only way to demonstrate the presence of recombinant sequences
is to examine the DNA itself, typically using a polymerase chain reaction (PCR) test.
[8]
Significant exceptions exist, and are discussed below.
If the rDNA sequences encode a gene that is expressed, then the presence of RNA
and/or protein products of the recombinant gene can be detected, typically using RTPCR or western hybridization methods.[8] Gross phenotypic changes are not the norm,
unless the recombinant gene has been chosen and modified so as to generate
biological activity in the host organism.[9] Additional phenotypes that are encountered
include toxicity to the host organism induced by the recombinant gene product,
especially if it is over-expressed or expressed within inappropriate cells or tissues.
In some cases, recombinant DNA can have deleterious effects even if it is not
expressed. One mechanism by which this happens is insertional inactivation, in which
the rDNA becomes inserted into a host cell's gene. In some cases, researchers use this

phenomenon to "knock out" genes to determine their biological function and importance.
[10]
Another mechanism by which rDNA insertion into chromosomal DNA can affect gene
expression is by inappropriate activation of previously unexpressed host cell genes.
This can happen, for example, when a recombinant DNA fragment containing an active
promoter becomes located next to a previously silent host cell gene, or when a host cell
gene that functions to restrain gene expression undergoes insertional inactivation by
recombinant DNA.

Applications of recombinant DNA technology[edit]

A group of GloFish fluorescent fish

Recombinant DNA is widely used in biotechnology, medicine and research. Today,

recombinant proteins and other products that result from the use of rDNA technology
are found in essentially every western pharmacy, doctor's or veterinarian's office,
medical testing laboratory, and biological research laboratory. In addition, organisms
that have been manipulated using recombinant DNA technology, as well as products
derived from those organisms, have found their way into many
farms, supermarkets,home medicine cabinets, and even pet shops, such as those that
sell GloFish and other genetically modified animals.
The most common application of recombinant DNA is in basic research, in which the
technology is important to most current work in the biological and biomedical sciences.
[8]
Recombinant DNA is used to identify, map and sequence genes, and to determine
their function. rDNA probes are employed in analyzing gene expression within individual
cells, and throughout the tissues of whole organisms. Recombinant proteins are widely
used as reagents in laboratory experiments and to generate antibody probes for
examining protein synthesis within cells and organisms. [2]
Many additional practical applications of recombinant DNA are found in industry, food
production, human and veterinary medicine, agriculture, and bioengineering. [2] Some
specific examples are identified below.
Recombinant chymosin

Found in rennet, chymosin is an enzyme required to

manufacture cheese. It was the first genetically
engineered food additive used commercially.
Traditionally, processors obtained chymosin from
rennet, a preparation derived from the fourth stomach of
milk-fed calves. Scientists engineered a non-pathogenic
strain (K-12) of E. coli bacteria for large-scale laboratory
production of the enzyme. This microbiologically
produced recombinant enzyme, identical structurally to
the calf derived enzyme, costs less and is produced in
abundant quantities. Today about 60% of U.S. hard
cheese is made with genetically engineered chymosin.
In 1990, FDA granted chymosin "generally-recognizedas-safe" (GRAS) status based on data showing that the
enzyme was safe.[11]
Recombinant human insulin
Almost completely replaced insulin obtained from animal
sources (e.g. pigs and cattle) for the treatment of
insulin-dependent diabetes. A variety of different
recombinant insulin preparations are in widespread use.
[12]
Recombinant insulin is synthesized by inserting the
human insulin gene into E. coli, or yeast
(saccharomyces cerevisiae)[13] which then produces
insulin for human use.[14]
Recombinant human growth hormone (HGH,
somatotropin)
Administered to patients whose pituitary glands
generate insufficient quantities to support normal growth
and development. Before recombinant HGH became
available, HGH for therapeutic use was obtained from
pituitary glands of cadavers. This unsafe practice led to
some patients developing CreutzfeldtJakob disease.
Recombinant HGH eliminated this problem, and is now
used therapeutically.[15] It has also been misused as a
performance enhancing drug by athletes and others.
[16]
DrugBank entry
Recombinant blood clotting factor VIII
A blood-clotting protein that is administered to patients
with forms of the bleeding disorder hemophilia, who are
unable to produce factor VIII in quantities sufficient to
support normal blood coagulation.[17] Before the
development of recombinant factor VIII, the protein was
obtained by processing large quantities of human blood
from multiple donors, which carried a very high risk of
transmission of blood borne infectious diseases, for
example HIV and hepatitis B.DrugBank entry

Recombinant hepatitis B vaccine

Hepatitis B infection is controlled through the use of a
recombinant hepatitis B vaccine, which contains a form
of the hepatitis B virus surface antigen that is produced
in yeast cells. The development of the recombinant
subunit vaccine was an important and necessary
development because hepatitis B virus, unlike other
common viruses such as polio virus, cannot be grown in
vitro. Vaccine information from Hepatitis B Foundation
Diagnosis of infection with HIV
Each of the three widely used methods for diagnosing
HIV infection has been developed using recombinant
DNA. The antibody test (ELISA or western blot) uses a
recombinant HIV protein to test for the presence
of antibodies that the body has produced in response to
an HIV infection. The DNA test looks for the presence of
HIV genetic material usingreverse transcription
polymerase chain reaction (RT-PCR). Development of
the RT-PCR test was made possible by the molecular
cloning and sequence analysis of HIV genomes. HIV
testing page from US Centers for Disease Control
(CDC)
Golden rice
A recombinant variety of rice that has been engineered
to express the enzymes responsible for carotenebiosynthesis.[9] This variety of rice holds
substantial promise for reducing the incidence of vitamin
A deficiency in the world's population.[18] Golden rice is
not currently in use, pending the resolution of regulatory
issues.
Herbicide-resistant crops
Commercial varieties of important agricultural crops
(including soy, maize/corn, sorghum, canola, alfalfa and
cotton) have been developed that incorporate a
recombinant gene that results in resistance to the
herbicide glyphosate (trade name Roundup), and
simplifies weed control by glyphosate application.
[19]
These crops are in common commercial use in
several countries.
Insect-resistant crops
Bacillus thuringeiensis is a bacterium that naturally
produces a protein (Bt toxin) with insecticidal properties.
[18]
The bacterium has been applied to crops as an
insect-control strategy for many years, and this practice
has been widely adopted in agriculture and gardening.
Recently, plants have been developed that express a

recombinant form of the bacterial protein, which may

effectively control some insect predators. Environmental
issues associated with the use of these transgenic crops
have not been fully resolved.[20]

History of
recombinant
DNA[edit]
The idea of recombinant DNA
was first proposed by Peter
Lobban, a graduate student of
Prof. Dale Kaiser in the
Biochemistry Department at
Stanford University Medical
School.[21] The first publications
describing the successful
production and intracellular
replication of recombinant
DNA appeared in 1972 and
1973.[22][23][24][25] Stanford
Universityapplied for a US
patent on recombinant DNA in
1974, listing the inventors
as Stanley N.
Cohen and Herbert W. Boyer;
this patent was awarded in
1980.[26] The first licensed drug
generated using recombinant
DNA technology was human
insulin, developed
by Genentech and Licensed
by Eli Lilly and Company.[27]

Controversy[edit]
Scientists associated with the
initial development of
recombinant DNA methods
recognized that the potential
existed for organisms
containing recombinant DNA
to have undesirable or
dangerous properties. At the
1975 Asilomar Conference on
Recombinant DNA, these

concerns were discussed and

a voluntary moratorium on
recombinant DNA research
was initiated for experiments
that were considered
particularly risky. This
moratorium was widely
observed until the National
Institutes of Health (USA)
developed and issued formal
guidelines for rDNA work.
Today, recombinant DNA
molecules and recombinant
proteins are usually not
regarded as dangerous.
However, concerns remain
about some organisms that
express recombinant DNA,
particularly when they leave
the laboratory and are
introduced into the
environment or food chain.
These concerns are discussed
in the articles on genetically
modified
organisms and genetically
modified food controversies.

See also[edit]
Biology portal
Molecular and cellular biology portal

Asilomar conference on
recombinant DNA

Genetic engineering

Genetically modified
organism

Recombinant virus

Vector DNA

Biomolecular engineering

Recombinant DNA
Technology

References[edit]
1.

Jump up^ Campbell, Neil A.

& Reece, Jane B..
(2002). Biology (6th ed.). San
Francisco: Addison Wesley.
pp. 375401. ISBN 0-20175054-6.

2.

^ Jump up to:a b c Peter

Walter; Alberts, Bruce;
Johnson, Alexander S.;
Lewis, Julian; Raff, Martin C.;
Roberts, Keith
(2008).Molecular Biology of
the Cell (5th edition,
Extended version). New York:
Garland Science. ISBN 08153-4111-3.. Fourth edition
is available online through
the NCBI Bookshelf: link

3.

Jump up^ Berg, Jeremy

Mark; Tymoczko, John L.;
Stryer, Lubert
(2010). Biochemistry, 7th ed.
(Biochemistry (Berg)). W.H.
Freeman &
Company. ISBN 1-42922936-5. Fifth edition available
online through the NCBI
Bookshelf: link

4.

^ Jump up to:a b Watson,

James D.
(2007). Recombinant DNA:
Genes and Genomes: A
Short Course. San Francisco:
W.H. Freeman. ISBN 0-71672866-4.

5.

Jump up^ Russell, David

W.; Sambrook, Joseph
(2001). Molecular cloning: a
laboratory manual. Cold
Spring Harbor, N.Y: Cold
Spring Harbor
Laboratory. ISBN 0-87969576-5.

6.

Jump up^ Hannig, G.;

Makrides, S. (1998).
"Strategies for optimizing
heterologous protein
expression in Escherichia
coli". Trends in
Biotechnology 16 (2): 54
60. doi:10.1016/S01677799(97)01155-4. PMID 948
7731. edit

7.

Jump up^ Brondyk, W. H.

(2009). "Chapter 11 Selecting
an Appropriate Method for
Expressing a Recombinant
Protein".Methods in
enzymology 463: 131
147. doi:10.1016/S00766879(09)63011-1. PMID 198
92171. edit

8.

^ Jump up to:a b c Brown,

Terry (2006). Gene Cloning
and DNA Analysis: an
Introduction. Cambridge, MA:
Blackwell Pub.ISBN 1-40511121-6.

9.

^ Jump up to:a b Ye, X.; AlBabili, S.; Klti, A.; Zhang, J.;
Lucca, P.; Beyer, P.;
Potrykus, I. (2000).
"Engineering the provitamin A
(beta-carotene) biosynthetic
pathway into (carotenoidfree) rice
endosperm". Science 287 (5
451): 303
305.doi:10.1126/science.287.
5451.303. PMID 10634784. e
dit

10. Jump up^ Koller, B. H.;

Smithies, O. (1992). "Altering
Genes in Animals by Gene
Targeting". Annual Review of
Immunology10: 705
730. doi:10.1146/annurev.iy.1
0.040192.003421.PMID 1591
000. edit
11. Jump up^ Donna U. Vogt
and Mickey Parish.
(1999) Food Biotechnology in
the United States: Science,
Regulation, and Issues

12. Jump up^ GualandiSignorini, A.; Giorgi, G.

(2001). "Insulin formulations-a review". European review
for medical and
pharmacological
sciences 5 (3): 73
83.PMID 12004916. edit
13. Jump up^ #Insulin aspart
14. Jump up^ DrugBank: Insulin
Regular (DB00030)
15. Jump up^ Von Fange, T.;
McDiarmid, T.; MacKler, L.;
Zolotor, A. (2008). "Clinical
inquiries: Can recombinant
growth hormone effectively
treat idiopathic short
stature?". The Journal of
family practice 57 (9): 611
612. PMID 18786336. edit
16. Jump up^ Fernandez, M.;
Hosey, R. (2009).
"Performance-enhancing
drugs snare nonathletes,
too". The Journal of family
practice58 (1): 16
23. PMID 19141266. edit
17. Jump up^ Manco-Johnson,
M. J. (2010). "Advances in
the Care and Treatment of
Children with
Hemophilia". Advances in
Pediatrics 57 (1): 287
294.doi:10.1016/j.yapd.2010.
08.007. PMID 21056743. edit
18. ^ Jump up to:a b Paine, J. A.;
Shipton, C. A.; Chaggar, S.;
Howells, R. M.; Kennedy, M.
J.; Vernon, G.; Wright, S. Y.;
Hinchliffe, E.; Adams, J. L.;
Silverstone, A. L.; Drake, R.
(2005). "Improving the
nutritional value of Golden
Rice through increased provitamin a content". Nature
Biotechnology 23(4): 482
487. doi:10.1038/nbt1082.PM
ID 15793573. edit
19. Jump up^ Funke, T.; Han,
H.; Healy-Fried, M.; Fischer,

M.; Schnbrunn, E.
(2006). "Molecular basis for
the herbicide resistance of
Roundup Ready
crops". Proceedings of the
National Academy of
Sciences 103 (35): 13010
13015.doi:10.1073/pnas.060
3638103. PMC 1559744.PMI
D 16916934. edit
20. Jump up^ Mendelsohn, M.;
Kough, J.; Vaituzis, Z.;
Matthews, K. (2003). "Are Bt
crops safe?". Nature
Biotechnology 21 (9): 1003
1009. doi:10.1038/nbt09031003.PMID 12949561. edit
21. Jump up^ Lear, J.
(1978). Recombinant DNA:
The Untold Story. New York:
Crown Publishers. p. 43.
22. Jump up^ Jackson, D.;
Symons, R.; Berg, P.
(1972). "Biochemical method
for inserting new genetic
information into DNA of
Simian Virus 40: Circular
SV40 DNA molecules
containing lambda phage
genes and the galactose
operon of Escherichia
coli". Proceedings of the
National Academy of
Sciences of the United
States of America 69 (10):
29042909. do
i:10.1073/pnas.69.10.2904. P
MC 389671.PMID 4342968. e
dit

23. Jump up^ Mertz, J. E.;

Davis, R. W.
(1972). "Cleavage of DNA by
R 1 restriction endonuclease
generates cohesive
ends".Proceedings of the
National Academy of
Sciences of the United
States of America 69 (11):
3370
4.doi:10.1073/pnas.69.11.33
70. PMC 389773.PMID 4343
968. edit

24. Jump up^ Lobban, P.;

Kaiser, A. (1973). "Enzymatic
end-to end joining of DNA
molecules". Journal of
Molecular Biology 78(3):
453471. doi:10.1016/00222836(73)90468-3.PMID 4754
844. edit
25. Jump up^ Cohen, S.;
Chang, A.; Boyer, H.; Helling,
R. (1973)."Construction of
biologically functional
bacterial plasmids in
vitro". Proceedings of the
National Academy of
Sciences of the United
States of America 70 (11):
3240
3244.doi:10.1073/pnas.70.11
.3240. PMC 427208.PMID 45
94039. edit
26. Jump up^ Hughes, S.
(2001). "Making dollars out of
DNA. The first major patent
in biotechnology and the
commercialization of
molecular biology, 19741980". Isis; an international
review devoted to the history
of science and its cultural
influences92 (3): 541
575. doi:10.1086/385281.PMI
D 11810894. edit
27. Jump up^ Johnson, I. S.
(1983). "Human insulin from
recombinant DNA
technology". Science 219 (45
85): 632
637.doi:10.1126/science.633
7396. PMID 6337396. edit

Further reading[edit]

Judson, Horace F.
1979. The Eighth Day of
Creation: Makers of the
Revolution in Biology.
Touchstone Books, ISBN
0-671-22540-5. 2nd
edition: Cold Spring
Harbor Laboratory Press,

1996 paperback: ISBN 087969-478-5.

Micklas, David. 2003. DNA

Science: A First Course.
Cold Spring Harbor
Press: ISBN 978-0-87969636-8.

Rasmussen,
Nicolas, Gene Jockeys:
Life Science and the rise
of Biotech Enterprise,
Johns Hopkins University
Press, (Baltimore),
2014. ISBN 978-1-42141340-2.

Rosenfeld, Israel.
2010. DNA: A Graphic
Guide to the Molecule that
Shook the World.
Columbia University
Press: ISBN 978-0-23114271-7.

Schultz, Mark and Zander

Cannon. 2009. The Stuff
of Life: A Graphic Guide to
Genetics and DNA. Hill
and Wang: ISBN 0-80908947-5.

Watson, James.
2004. DNA: The Secret of
Life. Random
House: ISBN 978-0-09945184-6.

External links[edit]
Library resources about
Recombinant proteins

Res
ources in your library

Res
ources in other libraries

Recombinant DNA fact

sheet (from University of
New Hampshire)

Plasmids in Yeasts (Fact

sheet from San Diego
State University)

Animation illustrating
construction of
recombinant DNA and
foreign protein production
by recombinant bacteria

Recombinant DNA
research at UCSF and
commercial application at
Genentech Edited
transcript of 1994 interview
with Herbert W. Boyer,
Living history project. Oral
history.

Recombinant Protein
Purification Principles and
Methods Handbook

[hide]

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The Basics of Recombinant DNA

So What Is rDNA?
That's a very good question! rDNA stands for recombinant DNA. Before
we get to the "r" part, we need to understand DNA. Those of you with
a background in biology probably know about DNA, but a lot of ChemE's
haven't
seen DNA since high school biology. DNA is the keeper of the all the
information
needed to recreate an organism. All DNA is made up of a base consisting
of sugar, phosphate and one nitrogen base. There are four nitrogen bases,
adenine (A), thymine (T), guanine (G), and cytosine (C). The nitrogen
bases are found in pairs, with A & T and G & C paired together. The sequence
of the nitrogen bases can be arranged in an infinite ways, and their structure is
known as
the famous "double helix" which is shown in the image below. The sugar used
in
DNA is deoxyribose. The four nitrogen bases are the same for all organisms.
The
sequence and number of bases is what creates diversity. DNA does not
actually make the organism, it only makes proteins. The DNA is transcribed
into mRNA and mRNA is translated into protein, and the protein then forms
the
organism. By changing the DNA sequence, the way in which the protein is
formed changes. This leads to either a different protein, or an inactive protein.

Now that we know what DNA is, this is where the recombinant comes in.
Recombinant DNA is the general name for taking a piece of one DNA, and
and combining it with another strand of DNA. Thus, the name recombinant!
Recombinant DNA is also sometimes referred to as "chimera." By combining
two or
more different strands of DNA, scientists are able to create a new strand of
DNA.
The most common recombinant process involves combining the DNA of two
different organisms.
How is Recombinant DNA made?
There are three different methods by which Recombinant DNA is made. They
are
Transformation, Phage Introduction, and Non-Bacterial Transformation. Each
are described separately below.

Transformation
The first step in transformation is to select a piece of DNA to be inserted
into a vector. The second step is to cut that piece of DNA with a restriction
enzyme and then ligate the DNA insert into the vector with DNA Ligase. The
insert contains a selectable
marker which allows for identification of recombinant molecules. An antibiotic
marker is often used so a host cell without a vector dies when exposed to a
certain
antibiotic, and the host with the vector will live because it is resistant.
The vector is inserted into a host cell, in a process called transformation. One
example of a possible host cell is E. Coli. The host cells must be specially
prepared to take up the foreign DNA.
Selectable markers can be for antibiotic resistance, color changes, or any
other
characteristic which can distinguish transformed hosts from untransformed
hosts.
Different vectors have different properties to make them suitable to different
applications. Some properties can include symmetrical cloning sites, size,
and
high copy number.
Non-Bacterial Transformation
This is a process very similar to Transformation, which was described above.
The
only difference between the two is non-bacterial does not use bacteria such as
E. Coli
for the host.
In microinjection, the DNA is injected directly into the nucleus of the cell being
transformed. In biolistics, the host cells are bombarded with high velocity
microprojectiles, such as particles of gold or tungsten that have been coated

with DNA.
Phage Introduction
Phage introduction is the process of transfection, which is equivalent to
transformation,
except a phage is used instead of bacteria. In vitro packagings of a vector is
used.
This uses lambda or MI3 phages to produce phage plaques which contain
recombinants.
The recombinants that are created can be identified by differences in the
recombinants and non-recombinants using various selection methods.

How does rDNA work?

Recombinant DNA works when the host cell expresses protein from the
recombinant genes.
A significant amount of recombinant protein will not be produced by the host
unless expression
factors are added. Protein expression depends upon the gene being
surrounded by
a collection of signals which provide instructions for the transcription and
translation
of the gene by the cell. These signals include the promoter, the ribosome
binding
site, and the terminator. Expression vectors, in which the foreign DNA is
inserted,
contain these signals. Signals are species specific. In the case of E. Coli,
these
signals must be E. Coli signals as E. Coli is unlikely to understand the signals
of
human promoters and terminators.
Problems are encountered if the gene contains introns or contains signals

which act
as terminators to a bacterial host. This results in premature termination, and
the recombinant
protein may not be processed correctly, be folded correctly, or may even be
degraded.
Production of recombinant proteins in eukaryotic systems generally takes
place in
yeast and filamentous fungi. The use of animal cells is difficult due to the fact
that many need a solid support surface, unlike bacteria, and have complex
growth
needs. However, some proteins are too complex to be produced in bacterium,
so eukaryotic cells must be used.

Why is rDNA important?

Recombinant DNA has been gaining in importance over the last few years,
and
recombinant DNA will only become more important in the 21st century as
genetic
diseases become more prevelant and agricultural area is reduced. Below are
some of the areas where Recombinant DNA will have an impact.

Better Crops (drought & heat resistance)

Recombinant Vaccines (ie. Hepatitis B)
Prevention and cure of sickle cell anemia
Prevention and cure of cystic fibrosis
Production of clotting factors
Production of insulin
Production of recombinant pharmaceuticals
Plants that produce their own insecticides
Germ line and somatic gene therapy

What does the future hold?

Now that we've figured out the basics behind what Recombinant DNA are, it's

time to look at how Recombinant DNA will impact the future. Which industries
and fields will be shaped by rDNA? How will rDNA effect the health and
lifestyles of RPI students in the next generation? Click over to our
rDNA Impact Statement to find out the answer!
Pop Quiz Time!
To help you determine how well you know Recombinant DNA, we
have generously decided to provide you with a basic quiz that even a
senior ChemE should be able to do. Be sure and look over the additional
information provided below, because these questions could be tricky! All
the information needed to answer the questions can be found on this page,
or the associated pages. When you're ready, click below.
Recombinant DNA Quiz

Additional Information
The information presented above is only an introduction to the wonders of
Recombinant DNA. In order to fulfill your desire for knowledge, Matt and
Beth have scoured the web for the best websites with in-depth knowledge
concerning rDNA. You will find the links below and a brief
description of what the page describes. Enjoy!
The URL

What you'll find

Recognition Sequences

Recognition Sequences for frequently used

restriction endonucleases.

Synthesized Human Proteins

Information about human proteins that have been

synthesized from eukaryotic and bacteria genes.

Gene Addition in Plants

Information about gene addition projects that have

been done with plants.

Gene Subtraction in Plants

Information about gene subtraction projects that

have been done with plants.

DNA Info

Basic information about what DNA is

DNA Replication

A SHOCKWAVE application illustrating DNA

replication

Protein Synthesis

A video that illustrates protein synthesis

Gene Splicing

Information about how gene splicing differs from

conventional agriculture
Gene Splicing

Information about the merits of agricultural gene

splicing

Genetic Diseases

Information about treating genetic diseases in the

womb

Gene Therapy

A Question and Answer about gene therapy

Recombinant DNA

The Recombinant DNA chapter of an online

textbook

Recombinant DNA
Technology

A Recombinant DNA problem set and tutorial

Recombinant DNA Research

The NIH Guidelines for research involving

Recombinant DNA

Recombinant DNA
Protocols

An online textbook covering the protocols for

Recombinant DNA

Clinical Trials

A clearinghouse of links concerning Clinical Trials

Gene Therapy

Information about gene therapy for human patients

Insulin Synthesis

Recombinant DNA and the synthesis of human

insulin

Medical Biotechnology

A repository of information concerning Medical

Biotechnology

Making Recombinant DNA

How does recombinant DNA technology work? The organism under study, which will be used to
donate DNA for the analysis, is called the donor organism. The basic procedure is to extract and
cut up DNA from a donor genome into fragments containing from one to several genes and allow
these fragments to insert themselves individually into opened-up small autonomously replicating
DNA molecules such as bacterial plasmids. These small circular molecules act as carriers,
or vectors, for the DNA fragments. The vector molecules with their inserts are

called recombinant DNA because they consist of novel combinations of DNA from the donor
genome (which can be from any organism) with vector DNA from a completely different source
(generally a bacterial plasmid or a virus). The recombinant DNA mixture is then used to
transform bacterial cells, and it is common for single recombinant vector molecules to find their
way into individual bacterial cells. Bacterial cells are plated and allowed to grow into colonies.
An individual transformed cell with a single recombinant vector will divide into a colony with
millions of cells, all carrying the same recombinant vector. Therefore an individual colony
contains a very large population of identical DNA inserts, and this population is called a DNA
clone. A great deal of the analysis of the cloned DNA fragment can be performed at the stage
when it is in the bacterial host. Later, however, it is often desirable to reintroduce the cloned
DNA back into cells of the original donor organism to carry out specific manipulations of
genome structure and function. Hence the protocol is often as follows:

MESSAGE

Cloning allows the amplification and recovery of a specific DNA segment from a large,
complex DNA sample such as a genome.
Inasmuch as the donor DNA was cut into many different fragments, most colonies will carry a
different recombinant DNA (that is, a different cloned insert). Therefore, the next step is to find a
way to select the clone with the insert containing the specific gene in which we are interested.
When this clone has been obtained, the DNA is isolated in bulk and the cloned gene of interest
can be subjected to a variety of analyses, which we shall consider later in the chapter. Notice that
the cloning method works because individual recombinant DNA molecules enter individual
bacterial host cells, and then these cells do the job of amplifying the single molecules into large
populations of molecules that can be treated like chemical reagents. Figure 10-1 on the following
page gives a general outline of the approach.

Figure 10-1

Recombinant DNA technology enables individual fragments of DNA from any genome to be
inserted into vector DNA molecules such as plasmids and individually amplified in bacteria.
Each amplified fragment (more...)
The term recombinant DNA must be distinguished from the natural DNA recombinants that
result from crossing-over between homologous chromosomes in both eukaryotes and
prokaryotes. Recombinant DNA in the sense being used in this chapter is an unnatural union of
DNAs from nonhomologous sources, usually from different organisms. Some geneticists prefer
the alternative name chimeric DNA, after the mythological Greek monster Chimera. Down
through the ages, the Chimera has stood as the symbol of an impossible biological union, a
combination of parts of different animals. Likewise, recombinant DNA is a DNA chimera and
would be impossible without the experimental manipulation that we call recombinant DNA
technology.
Go to:

Isolating DNA
The first step in making recombinant DNA is to isolate donor and vector DNA. General
protocols for DNA isolation were available many decades before the advent of recombinant
DNA technology. With the use of such methods, the bulk of DNA extracted from the donor will
be nuclear genomic DNA in eukaryotes or the main genomic DNA in prokaryotes; these types
are generally the ones required for analysis. The procedure used for obtaining vector DNA
depends on the nature of the vector. Bacterial plasmids are commonly used vectors, and these
plasmids must be purified away from the bacterial genomic DNA. A protocol for
extracting plasmid DNA by ultracentrifugation is summarized in Figure 10-2 on page 303.
Plasmid DNA forms a distinct band after ultracentrifugation in a cesium chloride
density gradient containing ethidium bromide. The plasmid band is collected by punching a hole
in the plastic centrifuge tube. Another protocol relies on the observation that, at a specific
alkaline pH, bacterial genomic DNA denatures but plasmids do not. Subsequent neutralization
precipitates the genomic DNA, but plasmids stay in solution. Phages such as also can be used

as vectors for cloning DNA in bacterial systems. Phage DNA is isolated from a pure suspension
of phages recovered from a phage lysate.

Figure 10-2

Plasmids such as those carrying genes for resistance to the antibiotic tetracycline (top left) can be
separated from the bacterial chromosomal DNA. Because differential binding of(more...)
Go to:

Cutting DNA
The breakthrough that made recombinant DNA technology possible was the discovery and
characterization of restriction enzymes. Restriction enzymes are produced by bacteria as a
defense mechanism against phages. The enzymes act like scissors, cutting up the DNA of
the phage and thereby inactivating it. Importantly, restriction enzymes do not cut randomly;
rather, they cut at specific DNA target sequences, which is one of the key features that make
them suitable for DNA manipulation. Any DNA molecule, from viruses to humans, contains
restriction-enzyme target sites purely by chance and therefore may be cut into defined fragments
of size suitable for cloning. Restriction sites are not relevant to the function of the organism, nor
would they be cut in vivo, because most organisms do not have restriction enzymes.
Lets look at an example: the restriction enzyme EcoRI (from E. coli) recognizes the following
sixnucleotide-pair sequence in the DNA of any organism:

This type of segment is called a DNA palindrome, which means that both strands have the
samenucleotide sequence but in antiparallel orientation. Many different restriction enzymes

recognize and cut specific palindromes. The enzyme EcoRI cuts within this sequence but in a
pair of staggered cutsbetween the G and the A nucleotides:

This staggered cut leaves a pair of identical single-stranded sticky ends. The ends are
called stickybecause they can hydrogen-bond (stick) to a complementary sequence. Figure 103 shows EcoRI making a single cut in a circular DNA molecule such as a plasmid: the cut opens
up the circle, and the linear molecule formed has two sticky ends. Production of these sticky ends
is another feature of restriction enzymes that makes them suitable for recombinant
DNA technology. The principle is simply that, if two different DNA molecules are cut with the
same restriction enzyme, both will produce fragments with the same complementary sticky ends,
making it possible for DNA chimeras to form. Hence, if both vector DNA and donor DNA are
cut with EcoRI, the sticky ends of the vector can bond to the sticky ends of a donor fragment
when the two are mixed.

Figure 10-3

The restriction enzyme EcoRI cuts a circular DNA molecule bearing one target sequence,
resulting in a linear molecule with single-stranded sticky ends.
MESSAGE

Restriction enzymes have two properties useful in recombinant DNA technology. First, they
cut DNA into fragments of a size suitable for cloning. Second, many restriction enzymes
make staggered cuts generating single-stranded sticky ends conducive to the formation of
recombinant DNA.
Dozens of restriction enzymes with different sequence specificities have now been identified,
some of which are shown in Table 10-1. You will notice that all the target sequences are
palindromes, but, like EcoRI, some enzymes make staggered cuts, whereas others make flush
cuts. Even flush cuts, which lack sticky ends, can be used for making recombinant DNA.

Table 10-1

Recognition, Cleavage, and Modification Sites of Various Restriction Enzymes.

DNA can also be cut by mechanical shearing. For example, agitating DNA in a blender will
break up the long chromosome-sized molecules into flush-ended clonable segments.
Go to:

Joining DNA
Most commonly, both donor DNA and vector DNA are digested with the use of a restriction
enzymethat produces sticky ends and then mixed in a test tube to allow the sticky ends of vector
and donor DNA to bind to each other and form recombinant molecules. Figure 10-4a shows
a plasmid vector that carries a single EcoRI restriction site, so digestion with the restriction
enzyme EcoRI converts the circular DNA into a linear molecule with sticky ends. Donor DNA
from any other source (say,Drosophila) also is treated with the EcoRI enzyme to produce a
population of fragments carrying the same sticky ends. When the two populations are mixed,
DNA fragments from the two sources can unite, because double helices form between their
sticky ends. There are many opened-up vector molecules in the solution, and many
different EcoRI fragments of donor DNA. Therefore a diverse array of vectors carrying different
donor inserts will be produced. At this stage, although sticky ends have united to generate a
population of chimeric molecules, the sugar-phosphate backbones are still not complete at two
positions at each junction. However, the backbones can be sealed by the addition of the
enzyme DNA ligase, which creates phosphodiester bonds at the junctions (Figure 10-4b). Certain
ligases are even capable of joining DNA fragments with blunt-cut ends.

Figure 10-4

Method for generating a chimeric DNA plasmid containing genes derived from foreign DNA.
(From S. N. Cohen, The Manipulation of Genes. Copyright 1975 by Scientific(more...)

Go to:

Amplifying Recombinant DNA

The ligated recombinant DNA enters a bacterial cell by transformation. After it is in the host cell,
theplasmid vector is able to replicate because plasmids normally have a replication origin.
However, now that the donor DNA insert is part of its length, the donor DNA is automatically
replicated along with the vector. Each recombinant plasmid that enters a cell will form multiple
copies of itself in that cell. Subsequently, many cycles of cell division will occur, and the
recombinant vectors will undergo more rounds of replication. The resulting colony of bacteria
will contain billions of copies of the single donor DNA insert. This set of amplified copies of the
single donor DNA fragment is the DNA clone (Figure 10-5 on the following page).

Recombinant DNA, which is often shortened to rDNA, is an artificially made DNA strand that
is formed by the combination of two or more gene sequences. This new combination may or
may not occur naturally, but is engineered specifically for a purpose to be used in one of the
many applications of recombinant DNA.
This article will go into further detail about what DNA is, how rDNA is made and what it can
be used for.

Background Information on DNA

DNA, also known scientifically as deoxyribonucleic acid, has a double helix structure and
contains a combination of the nitrogen bases: adenine, thymine, guanine and cytosine.
Although these four bases are the same in all organisms, they can be paired together and
arranged in an infinite number of ways, such that each organism has a unique combination
for their DNA strands.

Recombinant DNA
Recombinant DNA, or rDNA, is the term used to describe the combination of two DNA
strands that are constructed artificially. Genetic scientists can do this to create unique DNA
strand for different purposes, using several types of techniques.
Like naturally occurring DNA, recombinant DNA has the ability to produce recombinant
proteins. It is often these proteins that play the key role in the application of recombinant
DNA.

Formation of rDNA
In most cases, rDNA is created in a laboratory setting using a process of molecular cloning.
This method allows in vivo DNA replication, in the living cells of the subject.
A cloning vector is a DNA molecule that replicates inside a living cell and is used to form
rDNA. The cloning vector is usually a small part of a DNA strand that holds the genetic
information that is needed for the replication of cells. Polymerase chain reaction (PCR) is
another method that can be used to replicate a specific DNA sequence and create rDNA,
which is used to replicate DNA in a laboratory test tube.

The standard method of making recombinant DNA involves:

Choosing the appropriate host organism and cloning vector.

Preparation of vector DNA and DNA to be cloned.

Creation of recombinant DNA.

Introduction of rDNA to host organism.

Screening for rDNA with specific properties sought from host organisms.

Historical Overview
Peter Lobban and A. Dale Kaiser were the first scientists to propose a technique to form
recombinant DNA. This soon caught on to other scientists and in 1972 the first paper that
detailed a new way to insert genetic information using E. coli was released. Soon after in
1973, several other papers followed building on the concept and adding techniques of
construction and formation.
In 1978, Werner Arber, Daniel Nathans and Hamilton Smith were awarded the Nobel Prize in
Medicine for creating technology to discover, isolate and apply rDNA. Since this time,
recombinant genes and proteins have become widely used in medicine and agriculture. This
offers a novel method of managing some health conditions, such as the use of recombinant
insulin in diabetes, as well pest-control for gardens and farms.

References

http://www.ncbi.nlm.nih.gov/books/NBK21881/

http://ghr.nlm.nih.gov/handbook/basics/dna

http://www.cliffsnotes.com/sciences/biology/microbiology/dna-and-geneexpression/recombinant-dna-and-biotechnology