FUNDAMENTALS OF BIOTECHNOLOGY

FUNDAMENTALS OF
BIOTECHNOLOGY
Lecture # 08
Study!!
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Linkers & Adaptors
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Selection and Characterization of rDNA
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cDNA Libraries
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DNA transformation
Linkers and Adaptors
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Sticky ends are desirable for DNA cloning
experiments.
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Provided by treating the target and vectors with
same R.E or with different but producing the
sticky end.
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But some time target DNA blunt ended
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So therefore we will have to use Linkers and
Adaptors.
Linkers
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Synthetic , Short and known double stranded
oligonucleotides sequence.
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Having blunted ends on both sides and R. Sits.

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Treatment with R.E produces sticky ends after ligation
with target DNA.
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e.g. Linker having sit for BamHI.
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Drawback if target DNA also having the same R. Site
then?
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(Please use the book notes for detail).
Adaptors
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A Synthetic dstranded Oligonucleotide having
blunt end and Sticky end.
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Blunt ends will bind to the blunt ends of target
DNA to produce new DNA with sticky ends.
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Problems: sticky of adaptors will binds with each
other so….
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Treatment with Alkaline Phosphates.
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After attachment with target…… treatment
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Polynucleotide Kinase to add P–OH at 5 prime.
Homopolymer tailing (HT)
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Homopolymer: A strand composed of one type of

nucleotide.
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HT: the in-vitro addition of the same nucleotide by
the enzyme terminal deoxynucleotide transferase
to 3’-OH of a duplex DNA molecule. (calf
thymus).
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e.g. Complimentary poly (C) and poly (G) for
vector and target DNA respectively.
Selection and
Characterization of rDNA
Selection and Characterization of rDNA
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Identification and selection of rVector:
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Desirable antibiotic resistance
e.g. Ampicillline and tetracycline
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plaque formation:
X-gal
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Next step is to know which one contains our DNA of
interest.
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Genetic Method (Resistance, Color, plaque formation).
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Immunological screening: (inserted gene will produce
protein Ags and Abs reaction based)
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Hybridization method:
Prob will be hybridized with the corresponding DNA.
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DNA Sequencing:
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Southern and northern hybridization analysis
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Western blot for protein analysis
Screening and characterization of rClones
Selection with antibiotic resistance
(amp
r
)
Twin antibiotic resistance
Blue-white screening
Restriction digestion and Gel Electrophoresis
Hybridization to identify the interested
DNA or its RNA product
1. Radiolabeled probes which is complementary to
a region of the interested gene
Probes:

An oligonucleotide derived from the sequence
of a protein product of the gene

A DNA fragment/oligo from a related gene of
another species
1. Blotting the DNA or RNA on a membrane
2. Hybridize the labeled probe with DNA
membrane (Southern) or RNA (Northern)

membrane
Screening by Hybridization
Probes: DNA or RNA
100+ bp in size good
Sequence match >80% best
Stringency conditions
Screening Colonies by Hybridization
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Nucleic acid probe
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Cells transferred to
nylon membrane and
lysed
• DNA binds to
membrane, is denatured
and probe hybridized
• Bound probe detected
by autoradiography after
washing membrane
Screening by Immunological Assay
Screening by Functional Complementation
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Requires strain unable to
produce desired
product/function
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Cloned DNAs must be in
expression vector or
include elements required
for expression

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Select for restoration of
lost function
Southern and Northern blotting
Western blotting using a specific antibody
Identify the protein product of an interested
gene