Transcript Section J

Section J
Analysis and application of cloning
DNA
Section J -Analysis and application
of cloning DNA
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J1 CHARACTERIZATION OF CLONES
J2 NUCLEIC AND SEQUENCING
J3 POLYMERASE CHAIN REACTION
J4 ORGANIZATION OF CLONED
GENES
J5 MUTAGENESIS OF CLONED GENE
J6 APPLICATIONS OF CLONING
J1 CHARACTERIZATION OF
CLONES
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Characterization Determining various properties of
recombinant DNA molecule, such as size, restriction
map, orientation of any gene present and nucleotide
sequence, constitutes the process of clone
characterization. It requires a purified preparation of
the cloned DNA.
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Restriction mapping Digesting recombinant DNA
molecules with restriction enzymes, alone and in
combination, allows the construction of a
diagram(restriction map) of the molecules indicating
the cleavage position and fragment sizes.
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Partial digestion The partial digestion of endlabeled DNA fragments with restriction enzymes, and
sizing of fragments produced, also enables a restriction map to be
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Labelilg nucleic acid DNA and RNA can be endlabeled using polynucleotide kinase or terminal
transferase. Uniform labeling requires polymerases to
synthesize a complete labeled strand.
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Southern and Northern blotting The nucleic acid
in lanes of a gel is transferred to a memebrane, bound
and then hybridized with a labeled nucleic acid probe.
Washing removes nonhybridized probe, and the
membrane is then treated to reveal the bands
produced. Specific RNA species are detected on
Northern blots, whereas the DNA bands on Southern
blots could be genes in genomic DNA or parts of
cloned genes.
Fig. 1. Restriction mapping (a) using single and double digests to completion; (b) by partial
digestion of an end-labeled molecule. * is the labeled end
Fig. 2. 5′-End labeling of a nucleic acid
molecule.
Fig. 3. Southern
blotting.
J2 NUCLEIC AND
SEQUENCING
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DNA sequencing The two main methods of DNA
sequencing are Maxam and Gilbert chemical
method in which end-labeled DNA is subjected to
base-specific cleave reaction prior to gel separation,
and Snger’s enzymic method. The latter uses
dideoxynucleotides as chain terminators to produce
a ladder of molecules generated by polymerase
extension of primer.
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RNA sequencing A set of four Rnase that cleave
3’ to specific nucleotides are used to produce a
ladder of fragments from end-labeled RNA.
Polyacrylamide gel electrophoresis analysis allows
the sequence to be read.
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Sequence databases Newly determined
DNA ,RNA and protein sequences are entered
into databases(EMBL and GeneBank). These
collections of all known sequences are available
for analysis by computer.
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Analysis of sequences Special computer
software is used to search nucleic acid and
protein sequences for the presence of or
similarities
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Genome sequencing projects The entire
genome sequences of several organisms have
been determined and those of other organisms
are in progress. Often a genetic map is first
Fig. 1. DNA sequencing. (a) An example of a Maxam and Gilbert sequencing gel; (b)
Sanger sequencing.
J3 POLYMERASE CHAIN REACTION
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The PCR cycle The reaction cycle comprises a 95℃
step to denature the duplex DNA, an annealing step
of around 55℃ to allow the primers to bind and a
72℃ polymerization step. Mg2+ and dNTP are
required in addition to template, primers, buffer and
enzyme.
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Template Almost any source that contains one or
more intact target DNA molecule can, in theory, be
amplified by PCR, providing appropriate primers can
be designed.
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Primers A pair of oligonucleotides of about 18-30 nt
with similar G+C content will serve as PCR primers
as long as they direct DNA synthesis towards one
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Enzymes Thermostable DNA polymerases(e.g. Taq
polymerase) are used in PCR as they survive the hot
denaturation step. Some are more error-prone than
others.
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PCR optimization It may be necessary to vary the
annealing temperature and/or the Mg2+
concentration to obtain faithful amplification. From
complex mixtures, a second pair of nested primers
can improve specificity.
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PCR variation Variation on basic PCR include
quantitative PCR, degenerate oligonucleotide primer
PCR(DOP-PCR), inverse PCR, multiplex PCR, rapid
amplification of cDNA ends(RACE) and PCR
mutagenesis.
Fig. 1. The first three cycles of a polymerase chain reaction. Only after cycle 3 are there any duplex
molecules which are the exact length of the region to be amplified (molecules 2 and 7). After a few more
cycles these become the major product.
J4 ORGANIZATION OF CLONED
GENES
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Organization The polarity of oligo(dT)-primed cDNA clones
is often apparent from the location of the poly(A), and the
coding region can thus be deduced. The presence and polarity
of any gene in a genomic clone is not obvious, but can be
determined by mapping and probing experiments.
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Mapping cDNA on genomic DNA Southern blotting, using
probes from part of a cDNA clone, can show which parts of a
genomic clone have corresponding sequences.
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S1 nuclease mapping The 5’- or 3’- of a transcript can be
identified by hybridizing a longer, end-labeled antisense
fragment to the RNA. The hybrid is treated with nuclease S1
to remove single-stranded regions, and remaining fragment’s
size is measured on a gel.
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Primer extension A primer is extended by a polymerase until
the end of the template is reached and the polymerase
dissociates. The length of the extended product indicates the
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Gel retardation Mixing a protein extract with a
labeled DNA fragment and running the mixture on a
native gel will show the presence of DNA-protein
complexes as retarded bands on the gel.
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Dnase I footprinting The ‘footprint’ of a protein
bound specifically to a DNA sequence can be
visualized by treating the mixture of end-labeled
DNA plus protein with small amounts of Dnase I
prior to running the mixture on a gel. The footprint is
a region with few bands in a ladder of cleave products.
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Reporter genes To verify the function of promoter,
it can be joined to the coding region of an easily
detected gene(reporter gene) and the protein product
assayed under condition when the promoter should be
active.
DNA 3’
RNA 5’
* 5’
3’
Add S1 nuclease
RNA 5’
DNA 3’
3’
5’
PAGE Analysis
Fig. 1. S1 nuclease mapping the 5′-end of an RNA. * = position of end label.
Fig. 2. Primer extension. * = position of
end label.
J5 MUTAGENESIS OF CLONED
GENE
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Deletion mutagenesis Progressively deleting DNA from one
end is very useful for defining the important of particular
sequences. Unidirectional deletion can be created using
exonuclease III which removes one ztrand in a 3’ to 5’ direction
from a recessed 3’-end. A single strand –specific nuclease then
creates blunt end molecules for ligation, and transformation
generates then deleted clones.
Site-directed mutagenesis Changing one or a few nucleotides
at a particular site usually involves annealing a mutagenic
primer to a template followed by complementary strand
synthesis by a DNA polymerase. Formerly. Single-stranded
templates prepared using M13 were used, but polymerase chain
reaction(PCR) techniques are now preferred.
PCR mutagenesis By making forward and reverse mutagenic
primers and using other primers that anneal to common vector
sequences, two PCR reactions are carried out to amplify 5’and 3’-portions of the DNA to be mutated. The two PCR
Fig. 1. Unidirectional deletion mutagenesis
Fig. 2. PCR mutagenesis. X is the mutated site in the PCR product
J6 APPLICATIONS OF
CLONING
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Applications The various applications of gene
cloning incluse recombinant protein production,
genetically modified organism, DNA
fingerprinting, diagnostic kits and gene therapy.
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Recombinant protein By inserting the gene
for a rare protein into a plasmid and expressing
it in bacteria, large amounts of recombinant
protein can be produced. If post-translational
modifications are critical, the gene may have to
be expressed in aeukaryotic cell.
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Genetically modified organisms Imtroducting a foreign gene
into an organism which can propagate creats a genetically
modified organism. Transgenic sheep have been created to
produce foreign proteins in their milk.
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DNA fingerprinting Hybridizing Southern blots of genomic
DNA with probes that recognize simple nucleotide repeats
gives a pattern that is unique to an individual and can be used
as a fingerprint. This has applications in forensic science,
animal and plant breeding and evolutionary studies.
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Medical diagnosis The sequence information derived from
cloning medically important genes has allowed the design of
many diagnistic test kits which can help predict and confirm a
wide range of disorders.
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Gene therapy Attempts to correct a genetic disorder by
delivering a gene to patient are described as gene therapy.
Fig. 1. DNA fingerprinting showing how two VNTR alleles might be inherited (see text).
(a) Parental VNTR alleles. (b) Agarose gel analysis of VNTR alleles.