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