Transcript Chapter 3 part II

Recombinant DNA Technology
(Part II)
Genomic VS cDNA Library
• Size of the DNA fragments can be prepared
by different type of restriction enzymes.
• However, cDNA are of suitable size for
cloning without further manipulation.
• If one is interested in the amino acid
sequence of a protein – this information can
be obtained using cloned cDNA.
• If one is interested in the whole gene
including regulatory sequences, the genomic
DNA will be the suitable choice.
Making cDNA Library
• cDNA is the abbreviation for
complementary DNA or copy DNA
• A cDNA library is a set of clones
representing as many as possible of the
mRNAs in a given cell type at a given time
– Such a library can contain tens of thousands
of different clones
Making cDNA Library
• Isolation of poly(A) mRNA.
• Synthesis of cDNA by reverse transcription.
• cDNA molecules are joined to vector DNA to
create cDNA library.
• Screen library for desired cDNA clone.
Poly(A) mRNA isolation
• Isolate total RNA
• Bind mRNA to oligo(dT)
column
• Elute and discard rRNA
and tRNA
• Elute poly(A) mRNA
Making cDNA Library
• Central to successful cloning is the
synthesis of cDNA from an mRNA
template using reverse transcriptase (RT),
RNA-dependent DNA polymerase
– RT cannot initiate DNA synthesis without a
primer
– Use the poly(A) tail at 3’ end of most
eukaryotic mRNA so that oligo(dT) may serve
as primer
Making cDNA Library
• RT with oligo(dT) primer has made a
single-stranded DNA from mRNA
• Need to start to remove the mRNA
• Partially degrade the mRNA using
ribonuclease H (RNase H)
– Enzyme degrades RNA strand of an RNADNA hybrid
– Remaining RNA fragments serve as primers
for “second strand” DNA using nick translation
Making cDNA Library
 The nick translation process
simultaneously:
 Removes DNA ahead of a nick
 Synthesizes DNA behind nick
 Net result moves or translates
the nick in the 5’ to 3’ direction
 Enzyme often used is E. coli
DNA polymerase I
 Has a 5’ to 3’ exonuclease
activity
 Allows enzyme to degrade
DNA ahead of the nick
cDNA synthesis
• Reverse transcription
using oligo(dT) primer
linked with sequence
recognized by XhoI
• Nick RNA strand using
RNaseH
• Second strand cDNA
synthesis
Double stranded cDNA can
be modified to be cloned
into vector by adding
adapters or linkers
• Blunt 3’ overhang using
Pfu polymerase
• Add EcoRI adapter.
• Digest with XhoI.
• cDNA contains one end
compatible with EcoRI and
XhoI on the other end.
• Digest plasmid using
EcoRI and XhoI
• Ligate cDNA into
digested plasmids
• Transformation –
introduce recombinant
plasmids into bacterial host
cells.
• Select transformants
using blue-white
screening.
cDNA synthesis
• Reverse transcription
using oligo(dT) primer
• RT does not always
produce full length cDNA
• Nick RNA strand using
RNaseH
• Second strand cDNA
synthesis using T4 DNA
polymerase
Cloning full-length cDNA
1. Reverse transcription using oligo(dT) primer
linked with sequence recognized by restriction
endonuclease
2. RT synthesizes first strand of cDNA with 5methyl-dCTP
3. Biotin is attached to the end of mRNA
4. Rnase I degraded single stranded segments of
RNA
5. Full length RNA-DNA hybrid bind to streptavidin
Cloning full-length cDNA
6. Rnase H degrades the RNA of the RNA-DNA
streptavidin hybrid
7. A poly(dG) tail is added to the 3’ end
8. An oligo(dC) with sequence recognized by
second restriction enzyme is added
9. Second cDNA strand is synthesized
10. Final full length of cDNA is cloned into vector
Screening the Library
• Screening the library using nucleic acid
hybridization is the most direct and very sensitive
means for detecting the desired clones.
• This requires knowledge of the sequences of the
gene being sought.
• In some case, part of the gene may have already
been cloned, and this information can be used to
search for flanking sequence.
• Information might come from genome sequence
information of related organism.
DNA hybridization assay
• Double stranded DNA can be converted into
single stranded DNA by heat or alkaline
treatment. Heating breaks the H-bond but not
phosphodiester bond.
• If the heated solution is rapidly cooled, the strands
remain single stranded.
• If it is slowly cooled down, the helical
conformation of DNA can be established.
• This process is called annealing.
DNA hybridization assay
Random-primer method
The Klenow fragment
• Retains both DNA polymerase and 3’ exonuclease
activities but lacks of 5’ exonuclease activity.
• The 3’ exonuclease is retained because it reduces
the misincorporation of erroneous dNTPs during
the synthesis of new DNA strand.
• The 5’ exonuclease activity is abolished because
it would degrade some of the newly synthesized
DNA.
E. Coli DNA polymerase I
Random-primer method
Screening the Library
• From each discrete colony formed on a master plate,
a sample is transferred to a solid matrix, such as
nitrocellulose or nylon membrane.
• The cells on the membrane are lysed, and the
released DNA is denatured, deproteinized, and
irreversibly bound to the membrane (crosslinking).
• A labeled DNA probe is added to the membrane
under hybridization condition.
• After washing, exposing to an X-ray film, the colony
carrying the gene can be identified.
Southern Blot
Electrophoresis provides information on:
• Size of fragments. Fragments of known
size provide comparison.
• Presence of specific sequences. These
can be determined using probes.
DNA is denatured while in the gel, then
transferred to a nylon filter to make a
“blot.”
Identifying a specific clone with a
specific probe
 Probes are used to identify a desired clone from
among the thousands of irrelevant ones
 Two types are widely used
 Polynucleotides also called oligonucleotides
 Antibodies
Possible sources of probes
• First, cloned DNA from a closely related organisms (a
heterologous probe) can be used.
• Hybridization conditions need to be adjusted.
• Second, probe can be synthesized based on the
probable nucleotide sequence that is deduced from
the known amino acid sequence of the protein
encoded by the target gene.
Polynucleotide probes
Looking for a gene you want, might use homologous
gene from another organism
 If already cloned
 Hope enough sequence similarity to permit
hybridization
 Need to lower stringency of hybridization conditions to
tolerate some mismatches
Control of Hybridization Stringency
 Factors that promote separation of two strands
in a DNA double helix:
 High temperature
 High organic solvent concentration
 Low salt concentration
 Adjust conditions until only perfectly matched
DNA strands form a duplex = high stringency
 Lowering these conditions lowers stringency
until DNA strands with a few mismatches can
hybridize
Possible sources of probes
No homologous DNA from another organism?
 If amino acid sequence is known, deduce a set of
nucleotide sequences to code for these amino
acids
 Construct these nucleotide sequences chemically
using the synthetic probes
 Why use several?
 Genetic code is degenerate with most amino acids
having more than 1 nucleic acid triplet
 Must construct several different nucleotide
sequences for most amino acids
Screening by immunological assay
• From each discrete colony formed on a master plate,
a sample is transferred to a solid matrix, such as
nitrocellulose or nylon membrane.
• The cells on the membrane are lysed, and their
proteins are bound to the membrane.
• The membrane is treated with primary antibody that
binds only to the target protein.
• Unbound primary antibody is washed away, and the
membrane is treated with secondary antibody.
• Unbound secondary antibody is washed away and a
colorimetric is carried out to identify the clone.
Screening by protein activity
• From each discrete
colony formed on a
master plate, a sample
is transferred to a solid
matrix, such as
nitrocellulose or nylon
membrane.
Screening by functional
complementation
• Defective host cell (A-)
are transformed with
plasmids from genomic
library derived from
wildtype strain.
• The transformed cells
that carry a cloned gene
that confers the A+
function will grow on
minimal medium and
selected.
l Phage Vectors
 First phage vectors were constructed by Fred
Blattner and colleagues
 Removed middle region
 Retained genes needed for phage replication
 Could replace removed phage genes with foreign
DNA
 Originally named Charon phage
 More general term, replacement vectors
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Vectors for cloning large pieces
of DNA
 Phage vectors can receive larger amounts of foreign
DNA
 Charon 4 can accept up to 20kb of DNA
 Traditional plasmid vectors take much less
 Phage vectors require a minimum size foreign DNA
piece (12 kb) inserted to package into a phage
particle
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Cosmid Vectors
Cosmids are designed for cloning large DNA
fragments
 Behave as plasmid and phage
 Contain
 cos sites, cohesive ends of phage DNA that allow the DNA to be
packaged into a l phage head
 Plasmid origin of replication permitting replication as plasmid in
bacteria
 Nearly all l genome removed so there is room for large
inserts (40-50 kb)
 So little phage DNA can’t replicate, but they are infectious
carrying recombinant DNA into bacterial cells
Eukaryotic Vectors
 There are vectors designed for cloning genes into
eukaryotic cells
 Other vectors are based on the Ti plasmid to carry
genes into plant cells
 Yeast artificial chromosomes (YAC) and bacterial
artificial chromosomes (BAC) are used for cloning
huge pieces of DNA
Electroporation
• Cell suspension in
electroporation cuvette
• Cells and DNA in the
cuvette, prior, during,
and after high-voltage
electric field pulses
• Some cells acquire
exogeneous DNA
• Increase in
transformation
frequency
Tripartite mating
 Helper cell self-transfers a conjugative, mobilizing
plasmid with Tetr gene to a donor cell
 Donor cell contain nonconjugative, mobilizing
plasmid with Kanr gene
 Subsequently, nonconjugative, mobilizing plasmid
with Kanr gene is transported to a recipient cell
 Only recipient cell containing nonconjugative,
mobilizing plasmid with Kanr gene can be grown in
minimal medium with kanamycin