Transcript Principles of cell

Amplifying DNA: PCR & cellbased DNA cloning
1.
The importance of DNA cloning:
•
Current DNA technology is based on two different
approaches:
a. Specific amplification (DNA cloning) which involves
cell-based DNA cloning (involving a vector/replicon and
a suitable host cell) and in vitro DNA cloning (PCR)
b. Molecular hybridization where the DNA fragment of
interest is specifically detected using a mixture of
different sequences
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• 2. Polymerase Chain Reaction (PCR): features & Applications:
• Template DNA: DNA (linear or circular) or cDNA (complementary
DNA produced from produced mRNA by reverse transcriptase)
• Primers: pairs of oligonucleotides each 18-25 nucleotides long; 40%60% GC content; melting temp of both should not differ by >5oC; 3’
terminal sequences of any primer should not be to any sequences of the
other primer in the pair; self-complimentary sequences (inverted
repeats) of
>3 bp should be avoided.
• Cycling nature & exponential amplification: denaturation; primer
annealing; and DNA synthesis (extension).
• Regular Taq DNA polymerase lacks 3’ -> 5’ exonuclease activity
needed to provide proof-reading function.
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• PCR has two limitations:
a. short sizes of amplified products (<5 kb). This
is solved by doing Long-range PCR (up to tens of
Kb long) which uses a mixture of two heat stable
polymerases that provide optimal levels of DNA
synthesis as well as a 3’ -> 5’ exonuclease activity.
b. low yields of amplifications which is resolved
by cloning the PCR amplified DNA fragment in a
vector then propagating the vector in a cell based
system (clone by A/T cloning or by using
anchored PCR primers).
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• General Applications of PCR:
PCR has 3 major advantages:
- rapid
- sensitive
- robust (possible to amplify DNA from
damaged tissues or degraded DNA)
• Primer specificity is very important in PCR.
Several modifications have been developed to
reduce nonspecific binding (see Box 5.1):
- Hot-start PCR
- Nested PCR
- Touch-down PCR
• The correct base pairing at the extreme 3’ end of
bound primers is a requirement for producing a
PCR product. This allowed the use of PCR to
distinguish between alleles of the same gene that
differ in a single nucleotide (allele-specific PCR).
This method is known as ARMS (amplification
refractory mutation system).
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• Degenerate oligonucleotide primed PCR (DOPPCR) allow the amplification of a different but
closely related genes (novel genes) at the same
time.
•
Indiscriminate amplification of whole genomes
can be performed using linker-primed PCR
(ligation adaptor PCR).
• PCR could be used to amplify unknown DNA
sequences neighboring a known sequence. Such
methods include anchored PCR, inverse PCR,
RACE-PCR.
3. Principles of cell-based cloning:
• Four steps in cell-based cloning
- Construction of recombinant DNA molecules.
Involves the use of endonuclease restriction
enzymes, ligation, and a replicon (vector).
- Transformation in appropriate host cells.
- Selective propagation of cell clones. This step
takes advantage of selectable markers.
- Isolation of recombinant DNA from cell clones
followed by molecular characterization (such as
restriction enzyme analysis).
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• Endonuclease restriction enzymes type II (RE),
are a powerful tool (molecular scissors) used in
restricting target DNA (whole genome or plasmid)
into smaller DNA fragments. The restriction of a
DNA double helix molecule may result in a blunt
end or a cohesive end terminus (sticky end
generating a 3’ or 5’ single strand overhang). See
Table 5.1
• RE are used to generate recombinant linear
molecules (concatemers) or circular molecules
(cyclization).
• Simple cloning vectors include bacterial plasmids
and bacteriophages.
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• Recombinant DNA molecules are
transferred into appropriate host cells (e.g.
bacteria) for propagation. Normally a single
recombinant DNA exists per cell but
sometimes co-transformation may result in
two or more recombinant DNA molecules
per host cell.
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• DNA libraries are a collection of clones that
represent the entire genome of an organism.
Two types of libraries are known:
- Genomic library. To be representative of the
entire genome, the library should be >4GE. A
genome equivalent (GE) is genome size/average
insert size. In humans, for a GE=1, you need
3000Mb genome size)/40 kb (insert size) = 75,000
independent clones.
- cDNA library. Takes advantage of reverse
transcriptase. Usually much smaller than a
genomic library.
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• Selection of recombinant clones necessitates the
use of an appropriate selectable marker system.
- screening by vector molecules which includes
antibiotic resistance genes or β-galactosidase gene
complementation
- Generalized recombinant screening by
insertional inactivation. This can be achieved by
β-galactosidase screens or suppressor t-RNAbased screens.
- Directed recombinant screening. This can be
achieved by hybridization-based screening by
using labeled probes or by using PCR-based
screening.
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4. Cloning systems for different sized DNA
fragments:
• Such cloning systems normally include an
antibiotic resistance gene (to enable screening for
presence of vector) and a marker gene with a
multiple cloning site (to enable screening
recombinant clones).
• See Table 5.2 for different cloning vectors and the
DNA insert sizes that each could accommodate.
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• Lambda and cosmid vectors are used in cloning
moderately large DNA fragments in bacterial cells.
Three types of λ derived cloning vectors:
a. Replacement λ vectors: removal of central
section of the genome and replacing by a foreign
DNA fragment (up to 23 kb inserts)
b. Insertion λ vectors: modifications to allow
insertional cloning of cDNA fragments into the cI
gene (up to 5 kb)
c. Cosmid vectors: cos sequences of λ are inserted
into a small plasmid generating a cosmid. Can
take 33 – 44 kb inserts.
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• Bacterial Artificial Chromosome (BAC)
vectors are used to clone large fragments
(>300 kb). Low copy number (1-2
copies/cell) fertility factor (F-factor)
plasmids are used for this purpose.
• Bacteriophage P1 vectors and P1 artificial
chromosomes (PACs). Components of the
P1 phage are included in a circular plasmid
and can accommodate up to 122 Kb DNA
fragments.
• Yeast Artificial Chromosomes (YACs) permit the
cloning of 0.2 – 2.0 Megabases. YACs are
propagated in yeast as a linear chromosome which
becomes part of the genome and is distributed by
the mitotic machinery. YACs must include:
- centromere sequences (CEN)
- Telomere sequences (TEL)
- Autonomous replicating sequences (ARS) for
replication in the yeast nucleus.
- Ampicillin resistance for propagation in E. coli
- Three markers including a suppressor tRNA
gene, TRP1, and URA3 genes for selection by
complementation in the appropriate yeast host cell.
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5. Cloning systems for producing
mutagenized DNA:
• Cell-based oligonucleotide mismatch
mutagenesis can be used to generate a
specific nucleotide substitution in a coding
sequence of a gene. This is achieved by
using M13 vectors to generate singlestranded recombinant DNA
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• Production of single-stranded DNA for use
in sequencing is obtained using M13
vectors or phagemid vectors.
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• PCR-based mutagenesis could be used to achieve
two types of changes:
- 5’ adds-on mutagenesis which adds specific
sequences at the 5’ of the amplified product. Such
sequences may include a phage promoter to drive
gene expression.
- Site-directed mutagenesis which results in an
amplified product with a specific base substitution
to introduce a specific amino acid substitution at
the protein level.
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6. Cloning systems designed for gene
expression:
• Bacterial cells are used as hosts for
recombinant expression vectors designed for
the production of large amounts of a
recombinant protein (fusion proteins or tagged
proteins).
• Problems with overexpression in bacteria
include toxicity of large amounts of the
recombinant protein, lack of posttranslational
processing, inability to synthesize very large
mammalian proteins, and protein folding and
solubility.
• To solve the above mentioned problems:
- The use of pET-3 bacterial vectors containing T7
promoter in combination with host cells carrying the
gene for T7 RNA polymersae expressed under the
control of the lacZ promoter (i.e. inducible by IPTG).
- The vector is designed so that the recombinant protein
is fused to an endogenous protein (fusion proteins).
- Use an affinity tag so that the recombinant fusion
protein be purified by affinity chromatography. Two
affinity tagging systems are GST-glutathione affinity
and polyhistidine-nickel ion affinity.
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