recombinant dna technology (part ii)
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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. - PowerPoint PPT PresentationTRANSCRIPT
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 RNA-
DNA hybrid– Remaining RNA fragments serve as primers
for “second strand” DNA using nick translation
The nick translation process simultaneously:Removes DNA ahead of a nickSynthesizes DNA behind nickNet result moves or translates
the nick in the 5’ to 3’ direction
Enzyme often used is E. coli DNA polymerase IHas a 5’ to 3’ exonuclease
activity Allows enzyme to degrade
DNA ahead of the nick
Making cDNA Library
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 cDNA1. Reverse transcription using oligo(dT) primer
linked with sequence recognized by restriction endonuclease
2. RT synthesizes first strand of cDNA with 5-methyl-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 cDNA6. 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.”
Probes are used to identify a desired clone from among the thousands of irrelevant ones
Two types are widely usedPolynucleotides also called oligonucleotides Antibodies
Identifying a specific clone with a specific probe
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.
Looking for a gene you want, might use homologous gene from another organism
If already clonedHope enough sequence similarity to permit
hybridizationNeed to lower stringency of hybridization conditions to
tolerate some mismatches
Polynucleotide probes
Factors that promote separation of two strands in a DNA double helix:High temperatureHigh organic solvent concentrationLow 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
Control of Hybridization Stringency
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 tripletMust construct several different nucleotide
sequences for most amino acids
Possible sources of probes
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.
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Phage VectorsFirst phage vectors were constructed by Fred
Blattner and colleaguesRemoved middle regionRetained genes needed for phage replicationCould replace removed phage genes with foreign
DNAOriginally named Charon phageMore general term, replacement vectors
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Phage vectors can receive larger amounts of foreign DNACharon 4 can accept up to 20kb of DNATraditional plasmid vectors take much less
Phage vectors require a minimum size foreign DNA piece (12 kb) inserted to package into a phage particle
Vectors for cloning large pieces of DNA
Cosmids are designed for cloning large DNA fragments
Behave as plasmid and phageContain
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
Cosmid 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
Eukaryotic Vectors
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
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
Tripartite mating