Biology Score: Chapter 8: Recombinant Dna Technology
Biology Score: Chapter 8: Recombinant Dna Technology
Biology Score: Chapter 8: Recombinant Dna Technology
BIOLOGY
SCORE
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5’ 3’
G A A T T C
C T T A A G
3’ 5’
amp R
lacZ
4. Host Cell
A host cell is a cell that has been introduced
with DNA (or RNA), such as a bacterial cell acting as a host
cell for the DNA isolated from a bacteriophage. This cell is
utilized from the DNA cloning to accept, maintain and allow
the replication of cloning vector. This cell needs to have
these characteristics:
1. Able to receive DNA through the transformation
processes.
2. Able to maintain the structure of DNA recombinant
from one generation to other.
3. Able to amplify the gene product from the DNA
recombinant
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5. Modifying Enzymes
This is an enzyme used in the modification of DNA. For
example, DNA ligase. This enzyme catalyzed the formation of
phosphodiester bond between adjacent nucleotides in DNA.
Another example is, Taq polymerase. This is a DNA
polymerase isolated from thermophilic bacterium called
Thermus aquaticus. It catalyzes the addition of nucleotides to
DNA sequences. This enzyme is able to withstand high
temperature thus, it won’t be denatured.
BamHI 5’-GGATCC-3’
3’-CCTAGG-5’
SmaI 5’-GGGCCC-3’
3’-CCCGGG-5’
The list and The original plasmid is called a cloning vector, defined as a
explanation the DNA molecule that can carry foreign DNA into a host cell
types of cloning and replicate there. These are the examples of cloning
vectors.
vectors.
1) Plasmid
It is a small ring-shape DNA in bacteria (only a small
number of genes). It has the form of double stranded
circular DNA. However, it is not part of the bacteria’s
chromosome. But, it can self – replicating. For example,
pUC18
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2) Bacteriophage
Bacteriophage is a virus that infected bacteria. The genetic
information is in a linear form. It carries a larger DNA
inserts capacity then the bacteria. For example, λ2001
Genetic information
3) Cosmid
Cosmids can be used to build genomic libraries. Cosmids
can contain 37 to 52 kb of DNA, limits based on the
normal bacteriophage packaging size. They can replicate
as plasmids if they have a suitable origin of replication
(ori): for example SV40 ori in mammalian cells, ColE1 ori
for double-stranded DNA replication, They frequently
also contain a gene for selection such as antibiotic
resistance, so that the transformed cells can be identified
by plating on a medium containing the antibiotic. (Hybrids
of plasmids and bacteriophage lambda DNA). Other
example is SCOS-1
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4) YACs
Yeast artificial chromosomes (YACs) are genetically
engineered chromosomes derived from the DNA of the
yeast, Saccharomyces cerevisiae, which is then ligated into
a bacterial plasmid. By inserting large fragments of DNA,
from 100–1000 kb, the inserted sequences can be cloned
and physically mapped using a process called chromosome
walking. This is the process that was initially used for
the Human Genome Project, however due to stability issues,
YACs were abandoned for the use of Bacterial artificial
chromosomes (BAC). This chromosome is a double
stranded circular DNA. Other words, it is a plasmid with
yeast chromosome.
• Eg. pYAC
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BIOLOGY
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MAIN IDEAS
EXPLANATION NOTES
/KEY POINT
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• BUT, not all plasmid will join with the target DNA. This
caused two types of recombinant DNA:
Recombinant plasmid
Inserted gene of
interest
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5. Screening
The mixed bacteria are plate on nutrient-containing agar
medium supplemented with ampicillin and X-gal, a molecule
resembling lactose.
– Purpose: To identify bacterial colonies that carry
gene of interest / target DNA = Blue-white
screening.
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BIOLOGY
SCORE
MAIN IDEAS
EXPLANATION NOTES
/KEY POINT
Application Of • Many fields benefit from DNA technology and genetic engineering.
Recombinant - Agriculture
- Forensic
DNA
- Medicine – insulin for diabetics
Technology - Environment
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fore
but
t in
e.
47
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