Recombinant DNA Technology

Question 1

Also Genetic Engineering

Answer 1

Recombinant DNA Technology

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Refers to various techniques and procedures used in gene
manipulation

Answer 2

Recombinant DNA Technology

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Involves modifying and recombining DNAs to produce desired
products (e.g. proteins, or animals and plants with desirable
traits)

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Recombinant DNA Technology

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▪involves the use of molecular techniques to
modify the traits of target plant(s). The
resulting plants are often referred to as
transgenic plants or genetically modified
organisms (GMOs)

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Genetic engineering

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▪Achieved by adding a specific gene or genes
to a plant, or by knocking down a gene RNA,
to produce a desirable phenotype.

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Genetic engineering

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▪This gene can be from the same species or
different species organism

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Genetic engineering

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▪Sometimes genetic modification can produce
a plant with the desirable trait or traits faster
than classical breeding because the majority of
the plant’s genome is altered.

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Genetic engineering

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▪Practices involve focus on the mating of
organisms with desirable qualities. This relies
heavily on the naturally occurring plant life
cycle and homologous recombination to
generate genetic diversity and to eliminate
undesirable traits

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Classical engineering

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▪Involve the same species of plants.

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Classical engineering

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▪Interbreeding (crossing) can be only carried
out with closely or distant related plants.

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Classical engineering

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▪It is time-consuming as breeding needs
frequent crossing and self-fertilization.

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Classical engineering

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Tools used in Recombinant DNA
Technology

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• Target DNA
• Restriction enzyme
• DNA cloning vectors
• Host cell
• Modifying enzymes

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Also known as gene of interest

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Target DNA or gene

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A particular gene or DNA being studied
and manipulated in the experiment

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Target DNA or gene

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Identify a specific base sequence (restriction site) and cut
the DNA at specific point between two nucleotides in the
site

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Restriction Enzymes

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Different enzymes cleave at different base sequences.

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Restriction Enzymes

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Read 5’ to 3’ direction

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Restriction Enzymes

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Cut in staggered way resulting in DNA fragments with
unpaired bases at the end

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Restriction Enzymes

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are DNA molecules that carry foreign DNAs into
a host cell.

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Cloning vectors

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small, circular DNA molecules in bacteria - < 10kb
fragments
* Found in bacterial cell and in some eukaryotes
* Gene carried in plasmids provide bacteria with genetic advantages
(antibiotic resistant)

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Plasmid

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carries a foreign gene

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Recombinant plasmid

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linear double helix molecule (bacteria-infecting
viruses) - >10kb

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Bacteriophage

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are viruses that
infect and replicate within bacteria.

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Bacteriophages

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are highly specific to the type of bacteria they infect, making them potential tools for treating bacterial infections.

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Bacteriophages

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Characteristics of Cloning Vectors

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* able to accept foreign DNA in multiple cloning sites. * able to replicate freely and rapidly. * contain genes which are useful for bacteria (amp – code for antibiotic resistance)

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A cell which is able to accept foreign DNA (cloning vectors) and allow them to multiply.

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Host Cell

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Prokaryotic cell (E.coli) or eukaryotic cell (yeast or animal cell)

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Host Cell

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Characteristics of a Host Cell

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* Able to accept rDNA plasmids through transformation * Able to maintain the structure of rDNA from one generation to another * Able to amplify(multiply) the gene product of rDNA

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join fragments of DNA (target gene + plasmid = recombinant DNA)

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DNA ligase

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– replace DNA in PCR (polymerase chain reaction), a method for amplifying short segments of DNA

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Taq polymerase

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gene of interest

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Target DNA

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used to cut enzymes into fragments

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Restriction enzyme

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to carry target gene into a host cell (plasmid or bacteriophage)

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DNA cloning vectors

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bacterial cell that allows the cloning vectors to replicate with it

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Host cell

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DNA ligase and Taq polymerase

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Modifying enzymes

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Methods in Gene Cloning

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1. Isolation of the target gene 2. Insertion of the target gene into a vector 3. Introduction of the vector into a host 4. Amplification of the target gene by the host cell (cloning) and screening

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The desired gene is identified by:

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* by cutting the gene from a complete chromosome using a restriction enzyme and * by producing complementary

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fragments of various lengths

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DNA fragments

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reverse transcription

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DNA (cDNA)

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separate various fragments according to sizes

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Gel electrophoresis

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to locate the target gene

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DNA probe

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Insertion of the target gene into a vector

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* Target gene is inserted into vectors (plasmid or bacteriophage) resulting in recombinant DNA, combinations of DNAs from two sources. * The plasmids (opened rings) with sticky ends are mixed with the target gene and then joined by DNA ligase. * Results: plasmids that carry the target gene and/ or otherwise ( non-recombinant plasmid)

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Introduction of the vector into a host

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* The plasmids carrying the target gene (recombinant plasmids) must be introduced into a host cell. Results: * Bacteria that do not take up any plasmid * Bacteria that take up non-recombinant plasmid * Bacteria that take up recombinant plasmid

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Amplification of the target gene by the host cell (cloning) and screening

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* The bacteria is cultured in a medium containing ampicillin and the sugar, X-gal. * E. coli will be able to grow and form colonies (resistant to ampicillin) * The bacteria will divide and produce clones containing recombinant plasmids.

Question 40

A method of amplifying segments of DNA in vitro.

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Polymerase Chain Reaction (PCR)

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Polymerase Chain Reaction (PCR) Tools:

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Tools: thermocycler, a test tube with the DNA sample, Taq polymerase, primers and free DNA nucleotides

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Steps in PCR

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* Denaturation * Annealing * Elongation (Extension)

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The DNA is heated to break the hydrogen bond and separate the strands.

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Denaturation

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Each fragment is then cooled. - Add Taq polymerase and primers so they can bind to the ends of the DNA

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Annealing

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Add free DNA nucleotide and raise the temperature.

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Elongation (Extension)`

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Applied to the bacteria E. coli (Escherichia coli) to produce synthetic human insulin commercially

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Production of Insulin

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Transgenic animals – animals which genetically- modified

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Agriculture

Question 48

Transgenic plants – plants which genetically- modified