• Recombinant DNA is “an artificial DNA sequence resulting from the

combining of two other non relating DNA sequences in a plasmid”. It

is also referred to as rDNA.

• A plasmid is “ an extra nuclear fragment of DNA present in some

bacteria” .(Recombinant DNA, 2006)

• There are three methods of rDNA

– Transformation
– Phage induction
– Non-Bacterial (Kuure-Kinsey & McCooey, 2000)

• It is used for faster production of proteins, for the copying of genes,

and for giving an organism a new trait. (Blake et al, 2002)

• One of the DNA is usually a bacterial or viral DNA (vector DNA )

that accepts the other DNA molecule from an organism of interest.

• Results in the copying of many specific DNA segments (genes) which

code for a particular protein.

• rDNA is used in other biological applications such as gene therapy.

(Guilfoile, 2006)

• E. coli is the most common host cell used in bacterial recombinant

DNA. (Recombinant DNA, 2006)

• This presentation will focus on the transformation method of

recombinant DNA.
• It is commonly known as E. coli and belongs to
the family Enterobacteriacaea.
• This strain of bacteria was discovered by a
pediatrician and bacteriologist Theodor
Escherichia.
• E.Coli is usually found in the lower intestines of
warm blooded animals such as birds and
mammals. It is used in the proper digestion of
food.
• It is the ideal organism for the study of bacteria
because of its simple and clear structure.
• The fact that it is present in groundwater makes
it an indicator for fecal contamination.
• The average number of E.coli bacteria found in
the fecal matter excreted by a human in one day
is approximately 100 billion to 10 trillion.

(Escherichia Coli, 2006)

• First, a segment of DNA is selected from the original organism and inserted into a vector (ex. plasmid)

• Next, the gene (segment of DNA), that codes for a specific protein, is cut with a restriction enzyme called
restriction endonuclease at a region called the restriction (cleavage) site that is usually 4-6 nucleotides
long. The “cuts” are typically in the form of “sticky end” restriction segments.

• The DNA segment is then connected to the plasmid or the vector also using restriction endonuclease as
well as DNA ligase in a process called ligation. The sticky ends are paired with the corresponding
complementary bases
(Biotechnology, 2006/Recombinant DNA and Gene Cloning, 2006)

• This insert also contains a selectable marker used for identification of the recombinant molecule. It can
add characteristics such as antibiotic resistance and colour change to the host cell. (Kuure-Kinsey &
McCooey, 2000)

• The plasmid then enters the host cell (ex. E.coli) ,which is specially prepared to take in the foreign DNA,
where it uses the host cell’s enzymes for DNA replication. (Animated Tutorials: Molecular Biology)

• The plasmids, containing the DNA coding for particular proteins, are then produced by the bacteria
through bacterial replication
• The genes are then extracted (using restriction enzymes, gel-electrophoresis, sonication,etc…).
(Biotechnology, 2006)

• This process can be done in vitro or through PCR (Polymerase Chain Reaction)
(Kuure-Kinsey & McCooey, 2000)

• Phage Induction

• Another method of recombinant DNA is called phage induction.

• A process similar to transformation called transfection is used. The difference is that bacteriophages
(usually M13 or lambda) are used for the “in vitro packaging of a vector.” (Kuure-Kinsey & McCooey,
• Some bacteria can transfer a section of their chromosome to a organism
• That organism receives the chromosome through direct contact with the starting bacteria
or donor bacteria
• The bacteria copies the section of the transferring chromosome
• As the donor bacteria and the receiver connect the copy of the chromosome is injected
into it
• The transfer of the genes are continued until the two separate from each other
• The genes in the received from the donor replace the chromosomes or genes that are
similar in the recipients chromosomes.
(Kimball, 2006)
• “The occurrence of conjugation is due to the presence of certain plasmids in the donor
bacteria that posses genes for making the proteins involved in docking and transfer, and
then it is these plasmids that typically are what is transferred from one bacteria to the
other during the conjugative act” (Abedon, 2003)
• Restriction Fragments are: “small fragments of DNA that are cut from one DNA molecule
by a restriction endonuclease enzyme.” They are “ specific and predictable.”

• There are mainly two types of cuts; the staggered cut and a blunt cut.

• Staggered cuts are cuts done by a restriction enzyme that “leave a few unpaired
nucleotides remaining on a single strand at each end of a restriction fragment.”

• These unpaired nucleotides are called “sticky ends” and can link to complementary bases
in a DNA sequence.

(Blake et al, 2002)

Click to see animation on restriction endonuclease
(Animations-Restriction Endonucleases, 2005)
 Other Factors Involved
• DNA Amplification prepares for the rDNA process by “generating a large sample of a
target DNA sequence from a single gene or DNA fragment.” (Blake et al, 2002)
• rDNA works when the host cell expresses proteins from the recombinant genes.
(Kuure-Kinsey & McCooey, 2000)
• Proteins will not be generated by the host cell (ex.E.coli) unless expression factors
are added. Expression factors are: “plasmids that contain a prokaryotic promoter
sequence just ahead of a restriction enzyme target site” which are needed for the host
cell to transcribe the gene. (Blake et al, 2002)
• The signals generated by the expression factors are specific to different species
• Ex. “In E.coli these signals must be E.coli signals because E.coli will not understand
the signals of human promoters and terminators. Problems occur if the gene contains
introns or signals which act as the terminator to the host.”
• These problems result in premature termination and the recombinant protein may not
be processed/folded correctly. (Kuure-Kinsey & McCooey, 2000)
Click to watch a video of plasmid cloning

(Animated Tutorials: Molecular Biology)

• It is used for genetic transformation to produce genetically modified organisms.
(Recombinant DNA,2006)
• The Human Genome Project relied on this technology in order to provide a database of
genomic DNA molecules
• (Guilfoile, 2006)
• Better Crops (ex. resistant to heat, faster growing, bigger crops,etc…)
• Recombinant Vaccines (ie. Hepatitis B) (Kuure-Kinsey & McCooey, 2000)
• Prevention and cure of sickle cell anemia (erythropoitein (EPO)) (Recombinant DNA and
Gene Cloning, 2006)
• Prevention and cure of cystic fibrosis
• Production of clotting factors and prevention of blood clots
• Production of insulin
• Production of recombinant pharmaceuticals (Kuure-Kinsey & McCooey, 2000)
• To allow for the transplant of animal (pig) organs to the human body due to a shortage of
available human organs. Usually, the organs are rejected by the body. (Noonan, 2005)
• To produce “plant based” vaccines that are safe and effective. The process is cost-effective,
therefore a profit can be made by production companies. (Noonan, 2005)

Controversy

• Through the development of recombinant DNA techniques, concerns arose about the
potentially dangerous properties that could be created by genetically modified organisms
(Guilfoile, 2006)

• It has raised concern about the benefits and risks of developing genetically modified foods
using this technology. Altough it is cost-effective, the foods are not “natural”. (Guilfoile,
2006)
• Good
• Improves medicines by making it more
safe and cost effective to produce.
Improved livestock because of increased
resistance to disease and improved
crops lead to higher yields. (Kuure-
Kinsey & McCooey, 2000)
• Since 2001, more than 80 products used
for medicinal purposes have been
produced using this technology
• Better food products (ex. Rice with
higher nutritional values) (Guilfoile, 2006)
• Genetic diseases can be prevented.
• Helps to produce products such as:
insulin, growth hormones, oxytocin,
vaccines, etc.
• Treatment for pre-existing conditions for
cancer.
(Kuure-Kinsey & McCooey, 2000)
• Bad
• Safety and environmental concerns. “The
plasmids involved in the technology
account for the antibiotic resistant
strains of bacteria found in hospitals.”
(Recombinant DNA and Gene Cloning, 2006)
• Ethical dilemmas over human
treatments, people playing god. (Noonan,
2005)
• Potential experimental abuse.Patients
equal guinea pigs.
• Initially for germ line treatment in
treating disease, but now it is used to
pick traits for your child; abuse of the
technology. (Kuure-Kinsey & McCooey,
2000)
Insulin
Somatotropine-Human Growth Hormone

Rice Pigs containing organs that are potentially vital for human life.
 Bibliography
• Kuure-Kinsey,Matthew & Beth,McCooey M. (2000). The Basics of Recombinant DNA. Retrieved March 28,
2006, from An Introduction to Recombinant DNA Web site: http://www.rpi.edu/dept/chem-eng/Biotech-
Environ/Projects00/rdna/rdna.html.
• Recombinant DNA, the free encyclopedia. (2006). Retrieved March 30, 2006, from Recombinant DNA Web
site: http://en.wikipedia.org/wiki/Recombinant_DNA.
• Escherichia Coli. (2006). Retrieved March 28, 2006, from Escherichia Coli Web site:
http://en.wikipedia.org/wiki/Escherichia_coli.
• Guilfoile, Patrick G. "Recombinant DNA." Genetics. Ed. Richard Robinson. New York: Macmillan Reference
USA, 2003. Science Resource Center. Thomson Gale. 01 April 2006
<http://galenet.galegroup.com/servlet/SciRC?ste=1&docNum=CV2642650196>
• Recombinant DNA and Gene Cloning. (2006). Retrieved March 29, 2006, from Recombinant DNA and Gene
Cloning Web site: http://users.rcn.com/jkimball.mo.ultranet/ BiologyPages/
R/RecombinantDNA.html#Ligation_Possibilities..
• Biotechnology. (2006). Retrieved March 29, 2006, from Biotechnology Web site:
http://crystal.uah.edu/~carter/engineer.htm.
• Noonan, D. Newsweek.(2005). It's a gene pool party. , 145(26A), p.49,2p.
• Blake, Leesa, Craven,Meaghan, Dobell,Darcy, Flood,Nancy, Jasper,Gord,Little,Catherine,
Mason,Adrienne, Rrice,Grace. (2002). Mcgraw-Hill Ryerson Biology 12. Toronto, ONT: McGraw-Hill Ryerson.
• Animated tutorials: molecular biology. (n.d.). Retrieved Apr. 1, 2006, from Animation Development Web site:
http://www.sumanasinc.com/webcontent/anisamples/ molecular biology /molecularbiology.html.
• Animations-restriction endonucleases. (2005). Retrieved Apr. 1, 2006, from Animations Web site:
http://highered.mcgraw-hill.com/sites/0072437316/student_view0 /chapter16/ animations.html#.
• Kimball, J. W. (2006). Genetic recombination in bacteria. Retrieved Apr. 01, 2006, from Genetic Recombination in
Bacteria Web site: http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/Avery.html.
• Abedon , S. T. (2003). Recombinant dna and genetic engineering. Retrieved Apr. 01, 2006, from
Recombinant DNA and Genetic Engineering Web site: http://www.mansfield.ohio-
state.edu/~sabedon/black08.htm.