Cloning strategies

What are you trying to clone?

– cDNA or entire gene?

cDNA:

– used as probe to find gene in genomic library

– may be sequenced to find coding regions (exons)

– used for gene expression

entire gene:

– get complete gene sequence

– study expression/splicing etc. of eukaryotic gene

– in eukaryotic host

cloning cDNA:

– obtain mRNA, make cDNA copies

– clone in plasmid or lambda insertion vector

– get cDNA library

– screen library for desired cDNA

note – screening likely to be laborious or unsuccessful

– unless cDNA library is enriched for desired gene

– choose cell type/growth condition where gene expressed

– at higher level (if possible)

screening for cDNAs

– nucleic acid hybridization (see below)

– need specific (or enriched) probe

– expression screening (if cDNA in expression vector)

1. screen using specific antisera to protein

– most common technique

– prepare plaque lifts (for lambda insertion vectors)

– or colony lifts (for plasmid expression vectors)

– screen membrane with antisera

2. screen by complementation of mutants in host cell

– e.g. human genes cloned in yeast expression vectors

– got complementation of mutant yeast strains

– if human genes homologous to mutant yeast genes

3. screen by binding of protein to substrate

– e.g. identification of DNA-binding proteins

– “Southwestern” screening

– screen plaque lift/colony lift

– using labelled DNA probe

– get binding to any DNA

(e.g. histone clones)

– or binding to specific DNA sequence

– e.g. regulated promoter

– compare results

– find proteins that bind desired sequence

– note that denatured proteins will not bind DNA

– problem with E. coli expression systems

– one-hybrid screens

– similar to 2-hybrid screens

(for protein-protein interaction)

– but detect DNA-protein interaction instead

– clone ~3 copies of expected target DNA sequence

– upstream from regulated yeast marker gene

– e.g. HIS3 gene

– needed for histidine biosynthesis

– transcription requires activation domain

– must be bound to promoter

– by DNA-binding domain

– transform clone into HIS3 mutant yeast strain

– then prepare library of cDNA clones

– in activation domain fusion expression vector

– transform strain that has target/HIS3 construct

– if DNA-binding protein fused to activation domain

– get expression of HIS3 gene

– growth on minimal media

cloning entire genes

1. prepare genomic library

– screen with labelled nucleic acid probes

note: if location of gene known, may not need library

– if gene identified on specific BAC/YAC clone

– request from Genome Sequence Centre

– if linked to known gene (or DNA sequence)

– can do microdissection of metaphase chromosomes

– cut out part with gene – make smaller library

– if transposon mutant available:

– find gene using probe for transposon sequence

– or inverse PCR from ends of transposon

– also can use modified transposons – with marker genes

– screen for expression of marker gene – e.g. lacZ

2. or use PCR primer pair to amplify desired sequence

– requires PCR primers flanking gene

possible DNA probes:

1. specific cDNA for gene

– can be used to find genomic clone (if available)

What if specific cDNA not available?

– can enrich for mix containing proper cDNA

plus/minus screen – prepare 2 sets of 1^st strand cDNAs

– 1 from cell/condition where gene expressed (+)

– other from different cell/condition

– gene not expressed (–)

– radiolabel both cDNA pools

– use to screen duplicate plaque lifts

– prepared from libraries

– may screen genomic library for complete gene

– or cDNA library

– find clones that only hybridize to “+” cDNA pool

– study further

subtractive screening – prepare 2 sets of mRNAs

– 1 from cell/condition where gene expressed

– other from different cell/condition

– gene not expressed

– prepare 1^st strand cDNA from “+” pool

– degrade RNA in duplex

– hybridize cDNA pool with “–” mRNA pool

– separate DNA/RNA hybrids from unique “+” cDNAs

– using hydroxyapatite column

– binds double-stranded nucleic acids

(better than single-stranded)

– because of regular double-helix structure

– unique “+” cDNAs

– radiolabel & use as probes to screen libraries

– find genomic clone or cDNA clone

– good for finding rare mRNAs

– get rid of most mRNAs present

differential display – amplify mix of 3' ends of mRNAs

– different genes give different-sized cDNA products

– look for differences between “+” and “–” PCR products

– use unique “+”cDNAs as probes (as above)

Another form of enrichment:

RT-PCR or RACE

– amplify (part of) rare mRNAs by PCR

– use as probes to find genes or full cDNA clone

– need specific primers for gene

2. similar gene from related organism

– rely on conserved DNA sequences

3. oligonucleotides

– can be used as probe (1 oligo) or for PCR (pair)

– design oligos based on

– published genomic/cDNA sequence

– from same or related organism

– amino acid sequence of protein

– problem is degenerate genetic code

– can make “degenerate” oligo pools

– mix of different oligos

– for different possible sequence

– minimise number needed

– use sections of protein with:

Met, Trp – 1 codon each

Phe, Tyr, Cys, His, etc.

– 2 codons each

few Leu, Ser – 6 codons each

– or use I in probe

– binds to A, C, or T

– or use “guessmer”

– try to guess which codons will be in gene

– based on GC content, codon usage

(if known)