Recombinant DNA Technology

Question 1

Also Genetic Engineering

Answer 1

Recombinant DNA Technology

Question 2

Refers to various techniques and procedures used in gene
manipulation

Answer 2

Recombinant DNA Technology

Question 3

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

Answer 3

Recombinant DNA Technology

Question 3

▪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)

Answer 3

Genetic engineering

Question 3

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

Answer 3

Genetic engineering

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

Answer 4

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.

Answer 5

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

Answer 6

Classical engineering

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▪Involve the same species of plants.

Answer 7

Classical engineering

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

Answer 8

Classical engineering

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

Answer 9

Classical engineering

Question 11

Tools used in Recombinant DNA
Technology

Answer 11

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

Question 12

Also known as gene of interest

Answer 12

Target DNA or gene

Question 13

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

Answer 13

Target DNA or gene

Question 14

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

Answer 14

Restriction Enzymes

Question 15

Different enzymes cleave at different base sequences.

Answer 15

Restriction Enzymes

Question 16

Read 5’ to 3’ direction

Answer 16

Restriction Enzymes

Question 17

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

Answer 17

Restriction Enzymes

Question 18

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

Answer 18

Cloning vectors

Question 19

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)

Answer 19

Plasmid

Question 20

carries a foreign gene

Answer 20

Recombinant plasmid

Question 21

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

Answer 21

Bacteriophage

Question 21

are viruses that
infect and replicate within bacteria.

Answer 21

Bacteriophages

Question 22

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

Answer 22

Bacteriophages

Question 23

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)

Question 24

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

Question 26

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

Question 29

used to cut enzymes into fragments

Answer 29

Restriction enzyme

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

Answer 30

DNA cloning vectors

Question 31

bacterial cell that allows the cloning vectors to
replicate with it

Answer 31

Host cell

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DNA ligase and Taq polymerase

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Modifying enzymes

Question 33

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)

Question 38

Introduction of the vector into a host

Answer 38

• 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

Question 39

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

Answer 39

• 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.

Answer 40

Polymerase Chain Reaction (PCR)

Question 41

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

Question 42

Steps in PCR

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• Denaturation
• Annealing
• Elongation (Extension)

Question 43

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

Answer 43

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

Answer 44

Annealing

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Add free DNA nucleotide and
raise the temperature.

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Elongation (Extension)`

Question 46

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

Answer 46

Production of Insulin

Question 47

Transgenic
animals – animals which genetically-
modified

Answer 47

Agriculture

Question 48

Transgenic plants
– plants which

genetically-
modified