WHAT IS GENE CLONING ?

DNA cloning is a technique for reproducing DNA fragments. 

A vector is required to carry the DNA fragment of interest into the host cell.

DNA cloning allows a copy of any specific part of a DNA (or RNA) sequence to be selected among many others and produced in an unlimited amount.

IMPORTANCE OF GENE CLONING Mapping or sequencing of genomes cDNA sequences reveal expression

profiles in different cell types, developmental stages, and in response to natural or experimentally simulated external stimuli.

cDNA sequences provide useful information about splice isoforms and their abundance in different tissues and developmental stages.

Also reveal extra functional information about genes, e.g., expression profiles or biochemical functions.

SOURCE OF DNA SEGMENTS FOR CLONING

GENOMIC DNA cDNA

The cloning strategy is divided is divided into four stages :

a. Obtaining DNA fragmentsb. Joining to vectorsc. Introduction into host celld. Screening or Selection

Generalized overview of Cloning Strategies

• In cell-based cloning strategies, DNA fragments are initially generated and cloned in a non-specific manner, so that screening for the desired clone is carried out at the end of the process.

•When specific DNA fragments are obtained by PCR or direct chemical synthesis, there is no need for subsequent screening.

OBTAINING DNA FRAGMENTS

Mechanical shearing – the average fragment size can be controlled, but insertion of the resulting fragments into vectors requires additional modification steps

Restriction endonuclease digestion Duplex cDNA synthesis Direct chemical synthesis PCR

RESTRICTION ENDONUCLEASES Genomic DNA libraries are generated by

fragmenting the genome and cloning overlapping fragments in vectors.

The first genomic libraries were cloned in simple plasmid and phage vectors.

λEMBL series of vectors have been very widely used for genomic library construction. E.g. λ 2001, λDASH, EMBL3a

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Phage λ vectors v.s. High-capacity vectors Advantage of high-capacity vectors is

that the average insert size is much larger than for λ replacement vectors

The number of recombinants that need to be screened to identify a particular gene of interest is correspondingly lower, large genes are more likely to be contained within a single clone, and fewer clones are required to assemble a contig.

The main application of BAC, PAC and YAC libraries is for genome mapping, sequencing and the assembly of clone contigs.

PCR as alternative to genomic DNA cloning PCR with specific primers could be used to

isolate genes directly from genomic DNA, obviating the need for the production of genomic libraries.

Limitations of PCR……….. Suitable for the amplification of short products

– max. product size is 5kb. Poor processivity and proofreading of Taq

polymerase Extreme reaction conditions required which

cause damage to bases and generate nicks in DNA strands, increasing the probability of premature termination on long templates.

Duplex cDNA synthesis

cDNA is representative of the mRNA population.

Synthesis of double-stranded cDNA – First-strand DNA synthesis on the mRNA

template, carried out with a reverse transcriptase

Removal of the RNA template Second-strand DNA synthesis using the

first DNA strand as a template using DNAP

Significance of cDNA libraries The size of the cDNA clone is significantly

lower than that of the corresponding genomic clones

Eukaryotic cDNA clones find application where bacterial expression of the foreign DNA is necessary

As the sequence of the genomic DNA is available, the position of intron/exon boundaries can be assigned by comparison with the cDNA sequence

An important concept is that cDNA library is representative of the RNA population from which it was derived.

Abundance of a particular cDNA varies depending on cell type

Where a gene is differentially spliced, a cDNA library will contain different clones representing alternative splice variants

Hairpin method (Maniatis,1976)

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Okayama and Berg (1982)-Directional cDNA cloning

PCR as an alternative to cDNA cloning Reverse transcription followed by the

polymerase chain reaction leads to the amplification of RNA sequences in cDNA form.

The sensitivity is such that total RNA can be used as the starting material, rather than the poly(A) RNA which is used for conventional cDNA cloning.

Its pros and cons compared to cDNA library………. Screening a cDNA library is

laborious; cDNA library may not yield a cDNA clone with a full-length coding region

Specialized cell library act as reference for PCR amplification

cDNA library may be used as a source from which a specific cDNA can be obtained by PCR amplification

• PCR based screening strategies are dependent upon specific primers; with cDNA libraries screening strategies are based upon expression•The DNA polymerases used for PCR are more error-prone than those used conventionally for second-strand synthesis, so the library may contain a large number of mutations•False results from RT-PCR based cDNA library construction as a result of the amplification of contaminating genomic sequences in the RNA preparation

Rapid amplification of cDNA ends (RACE) – for full length cDNA cloning

Rapid Amplification of cDNA Ends – PCR (RACE - PCR) permits cloning of 5' or 3' ends of a specific, target cDNA, and not all cDNAs. Prior protein purification and sequencing permits the use of RACE-PCR to selectively amplify the cDNA sequence of the desired protein, unlike RT-PCR, which amplifies all cDNA sequences.

The steps to perform RACE-PCR are:1. All mRNA transcripts are reverse transcribed (RT) into cDNAs. 2. Then using primers specific for the target cDNA, separate PCR reactions are run to individually clone each end of the target cDNA. 3. The PCR products are sequenced, new primers are designed, and the entire target cDNA is cloned.

RACE-PCR requires a knowledge of the target protein’s amino acid sequence, which will enable the design of primers specific for the target gene. The advantage of RACE-PCR over RT-PCR or PCR gene cloning strategies is that if one successfully copies the correct, full – length gene sequence, there won’t be a need to construct and screen a cDNA library, thereby saving a lot of time and labour.

CAPture method of full-length cDNA cloning First strand cDNA synthesis is

initiated as usual, using an oligo-dT primer.

Formation of full-length and partial length cDNA fragments

RNase A treatment Isolation on eIF-4E affinity column

and subsequent elution of full length cDNA

Oligo-capping – for full length cDNA

SCREENING STRATEGIES Screening of genomic/cDNA libraries for

clones is done by:• Nucleic acid hybridization• PCR

Screening of product of a clone applies only to expression libraries and is done by:

• Immunological screening• Southwestern and northwestern screening • Functional cloning• Positional cloning• Differential screening

Nucleic acid hybridization It is the most commonly used method of

library screening because it is rapid, it can be applied to very large numbers of clones, and in the case of cDNA libraries, can be used to identify clones that are not full length.

Colony hybridization – Grunstein and Hogness (1975) – in situ hybridization using radioactive RNA probes into transformed colonies for detection of DNA sequence

Plaque lift technique – Benton and Davis (1977)

Immunological screening

Involves the use of antibodies that specifically recognize antigenic determinants on the polypeptide synthesized by a target clone

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Southwestern and Northwestern screening

Used for screening and isolation of clones expressing sequence-specific nucleic acid binding proteins.

In Southwestern blotting, screening is done by incubating the membranes with a radiolabeled double-stranded DNA oligonucleotide probe, containing the recognized sequence for the target DNA-binding protein.

• Northwestern blotting is used to isolate sequence-specific RNA-binding proteins, using a single-stranded RNA probe

Functional cloning/Functional complementation Screening method that depend on the full

biological activity of the protein. A particular DNA sequence compensates

for a missing function in a mutant cell, and thus restores the wild-type phenotype.

If mutant cells are non-viable (auxotrophic mutations) under particular growth conditions, cells carrying the clone of interest can be positively selected, allowing the corresponding clone to be isolated.

Positional cloning

It is used when there is no biological information about a gene, but its position can be mapped relative to other genes or markers

A technique known as chromosome walking is used, wherein overlapping clones are obtained spanning the region of interest by using each successive clone as a probe to detect the next

Differential screening

This is a variation of DNA hybridization method that is suitable for isolating tissue-specific cDNA sequences

e.g. The isolation of cDNA derived from mRNA which are abundant in gastrula embryo from frog but not present in the egg.

1. cDNA clone library prepared from gastrula mRNA

2. Replica sets of recombinant clones prepared 3. One filter probed with radiolabelled mRNA or

cDNA from gastrula embryos 4. One filter probed with radiolabelled cDNA or

mRNA from egg

some colonies will give positive signal with both probes

             = cDNA from mRNA abundant in both stages of development

some colonies will not give positive signal with either probe

            = mRNA of low abundance in both stages of development

some colonies give positive signal with only gastrula probe but not egg probe

        = correspond to required sequence