BIO4320 Lecture Materials, Prepared by Dr.

Hon-Ming Lam

Construction and Screening of Gene Libraries

Further Readings: Genome II by TA Brown, Ch. 4; Current Protocols in Molecular Biology by Ausubel et al., Ch. 5 and 6; PNAS 88:1731-35

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Tissue/Cell mRNA cDNA

(methylation; addition of linkers, etc.)

Vectors DNA
(cDNA: plasmids, phage Genomic: phage, cosmid, BAC, PAC, YAC, TAC)

Partial or Complete Restriction Restriction Ligation Transformation, in vitro packaging, etc

Size Fractionation

Screening for desired clones

Amplify for longterm storage

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Genomic Libraries
from genomic DNA frequency of hits independent of gene expression levels

cDNA Libraries
reverse transcription of mRNA dependent

may contain promoters and no promoters or introns introns expression is feasible if linked cannot express in to a suitable promoter heterologous system useful for genome analysis, useful for analysis of coding regions and gene functions map-based cloning, promoter studies, etc

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Representation and Randomness

Q = (1 1/n)N Q: probability of a clone not in a random library P: probability of including any DNA sequence in a random library N: independent recombinant clones to be screened

P=1Q P = 1 (1 1/n)N (1 1/n)N = 1 P

n: total number of clones needed to ln (1 1/n)N = ln (1 P) cover the total genome if the clones are not overlapping (total size of genome divided by the N = ln (1-P) / ln (1-1/n) average size of a single cloned fragment)

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Representation and Randomness

Genomic Libraries
N = ln (1 P) / ln (1 1/n) P: probability of including any DNA sequence in a random library N: independent recombinant clones to be screened n: total number of clones needed to cover the total genome if the clones are not overlapping (total size of genome divided by the average size of a single cloned fragment)

cDNA Libraries
N = ln (1 P) / ln (1 m/T) P: probability that each mRNA will be represented once N: independent recombinant clones to be screened m: number of molecules of the rarest mRNA in a cell T: total number of mRNA molecules in a cell

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Representation and Randomness

Genomic Libraries
N = ln (1 P) / ln (1 1/n) To have 99% chance of getting a desired sequence, screen 4.6 times the total number of base pairs

cDNA Libraries
N = ln (1 P) / ln (1 m/T)
Since it is hard to estimated the total number of mRNA molecules and the number of rarest mRNA molecules, it is difficult to predict the exact number of clones to be screened. In general, to have at least one copy of every mRNA, 500,000 to 1,000,000 independent cDNA clones should be screened

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Vectors for Genomic Libraries

More details can be found here: http://batzerlab.lsu.edu/Hum_Mol_Gen_Lectures/Hum%20Mol%20Genet%20Lecture%203%20Feburary%203,%202004.ppt

Vector

Insert Size

Remarks

YAC (yeast 230 - 1700 kb Propagate in Saccharomyces cerevisiae; artificial (length of Three major elements: chromosome) natural yeast centromere for nuclear division; chromosome) telomeres for marking the end of Average: 400the chromosome; origins of replication for initiation of new 700 kb
DNA synthesis when the chromosome divides Previously an important tool to map complex genomes; Problems: chimera, instability (rearrangement)

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Vectors for Genomic Libraries

Vector BAC (bacterial artificial chromosome) Bacteriophage P1 Insert Size Remarks Up to 300 kb Plasmid vector containing the F factor replicon; Average: One copy per bacterial cell 100 kb Maximum Deletion version of a natural phage genome about 100 kb P1 phage genome is about 110 kb Efficient packaging system pac cleavage site for recognition P1 plamsid replicon and inducible P1 lytic replicon loxP site for Cre action Similar to BAC Similar to P1 A combination of BAC and P1 features

PAC (P1derived artificial chromosome) TAC (Transformable artificial chromosome)

With P1 plasmid replicon (single copy in E. coli) and Ri plasmid replicon (single copy in Agrobacterium tumefaciens) With T-DNA border and can transform plant directly

Sources: www.genetics.wisc.edu/courses/spring04/466/files/bacteria2.pdf

Sources: www.genetics.wisc.edu/courses/spring04/466/files/bacteria2.pdf

Sources: http://batzerlab.lsu.edu/Hum_Mol_Gen_Lectures/Hum%20Mol%20Genet%20Lecture%203%20February%201,%202005.ppt

P1 vector for the construction of recombinants by P1 packaging

loxP P1 plasmid replicon Ad2 Sca 1 pBR322 ori pac loxP sacB Sp6 T7 sacB Sfi 1 S loxP pac Pierce et al, Proc. Natl. Acad. Sci.U.S.A. (1992) 89, 2056-2060 B B BamH 1 Not 1 kan c 1 repressor binding site loxP S P1 lytic replicon E.coli promoter

pAD10SacBII (30kb)
kan

Sources: http://batzerlab.lsu.edu/Hum_Mol_Gen_Lectures/Hum%20Mol%20Genet%20Lecture%203%20February%201,%202005.ppt

Sources: http://batzerlab.lsu.edu/Hum_Mol_Gen_Lectures/Hum%20Mol%20Genet%20Lecture%203%20February%201,%202005.ppt

Constructing P1 recombinants using packaging extracts

S pac loxP insert of 80-90 kb B B kan loxP S

packaging

loxP

kan

loxP

site-specific recombination

loxP

Sources: http://batzerlab.lsu.edu/Hum_Mol_Gen_Lectures/Hum%20Mol%20Genet%20Lecture%203%20February%201,%202005.ppt

P1 Derived Artificial Chromosomes (PACs)

Electroporation Based System
Large insert size Low copy number origin for propagation High copy origin for DNA production Negative selection against non-recombinants Very stable inserts

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Vectors for Genomic Libraries

Vector Insert Remarks Size Phages Up to Genome size of phages is about 47 kb; 20-30 Packaging system is efficient and can kb handle a total size of 78-105% of the genome; Replacement vector system is usually employed; Pre-digested arms are commercially available for library constructions; Useful for study of individual genes

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Vectors for Genomic Libraries

Vector Cosmid Insert Size 35-45 kb Remarks Plasmid contain the cos site of phage and hence can use phage packaging system; Propagate in E. coli as plasmids; Useful for subcloning of DNA inserts from YAC, BAC, PAC, etc. Contain F plasmid origin of replication and cos site; Low copy number and hence more stable

Fosmids

Similar to cosmid

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Vectors for cDNA Libraries

Vector
Phages

Insert Size
Up to 20-30 kb (for replacement vectors) and 10-15 kb (for insertion vectors)

Remarks
Maximum size for mRNA is about 8 kb, hence the capacity of DNA insert is not a major concern here; Insertion vector system is usually employed; Useful for study of individual genes and their putative functions Efficient packaging system, easy for gene transfer into E. coli cells, more representative than plasmid libraries, subcloning and subsequent DNA manipulation processes are less convenient than plasmid systems Relatively easy to transform E. coli cells although may not be as efficient as the phage system for large scale gene transfer; Less representative than phage libraries, subcloning and subsequent DNA manipulation processes are more convenient than the phage systems

Bacterial Up to 10-15 plasmids kb

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Combining the Advantage of the Phage and Plasmid Systems

Embed a plasmid vector in a vector In vitro package, transfect, propagate, and screen as phages Excise the plasmid for the vector and propagate and manipulate as plasmids subsequently, e.g. Excise by filamentous helper phages (e.g. Strategene) Excise by the Cre-lox system (Clontech)

ZAP Library (Stategene)

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

First Strand Synthesis

mRNA AAAAAAAA TTTTTTTTGAGCTC 1st Strand CH CH 3 3 cDNA CH3 + Linker-primer Reverse transcriptase (no RNase activities) 5-methyl dCTP, dATP, dGTP, dTTP

linker-primer: reverse transcription, restriction site (XhoI) 5-methyl dCTP: protect internal sites

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Second Strand Synthesis

2nd Strand cDNA AAAAAAAACTCGAT TTTTTTTTGAGCTC CH3 CH3 CH3 + RNase H and DNA polymerase I; dNTPs
RNase H: remove mRNA in DNA-RNA hybrid DNA Polymerase I: synthesis 2nd strand Both enzymes added simultaneously When RNase H starts to degrade the mRNA, residual RNA fragments may act as primers for initiation of DNA synthesis DNA Polymerase 1 fills the gaps by nick translation

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Addition of Adapter
+ EcoRI adapter; ligase
AATTC... G... CH3 CH3 CH3 AAAAAAAACTCGAT... G TTTTTTTTGAGCTC... CTTAA

+ XhoI
AATTC... G... CH3 CH3 CH3 AAAAAAAAC TTTTTTTTGAGCT

EcoRI Adapter and XhoI linker: directional cloning

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Primary Library Synthesis

Size fractionation Ligation to arms In vitro package (packaging extract should be McrA-, McrB-, and Mrr- to prevent digestion of hemimethylated DNA) Host for phage infection, e.g. XL1-Blue MRF:
Restriction deficient (mcrA)183, (mcrBC-hsdSMR-mrr)173; supE to allow propagation of helper filamentous phages (with amber mutation); recA- to prevent recombination F plasmid: M15lacZ for blue-white screening lacIq to prevent uncontrolled expression of fusion protein F pili to allow infection of helper filamentous phages

ZAP Library (Stategene)

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Excision of pBluescript Plasmid

First host XL1-Blue MRF: Fplasmid provides F pili for f1 filamentous phage attachment; it is a suppressing strain so that f1 helper phages which carry an amber mutation can pack DNA The ZAP: contains initiation and termination signal sequences for f1 packing flanking pBluescript sequence Second host: SOLR, a non-suppressing strain to stop helper phage propagation; a phage resistant strain to prevent phage propagation

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

XL1-Blue MRF

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

XL1-Blue MRF

F Co-transfection of ZAP and fl help phage

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

XL1-Blue MRF T I

F The ZAP phage clone replicates in the host cell The fl helper phage protein nicks the fl replication initiation site I on ZAP and replication continues until reaching the fl replication termination site T

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

XL1-Blue MRF

F fl phage coats indiscriminately pack DNA molecules containing the fl replication origin; both fl genome and pBluescript fragments are packed

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

XL1-Blue MRF

F fl phages are secreted to the growth medium

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

SOLR

F fl phages infect a new host SOLR (R, suppressor free)

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

SOLR

F fl phages infect a new host SOLR (R, suppressor free)

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

SOLR

F fl genome (contains an amber mutation) cannot propagate in the suppressor free SOLR and pBluescript containing cells can be selected by the ampicillin resistance marker

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Screening for the Right Clones

By PCR or hybridization if DNA sequence information of the target gene or homologous genes, or the amino acid sequence of the target gene product is available By hybridization if DNA fragments of the target gene or homologous genes are available By functional assays, e.g. Yeast functional complementation Microinjection

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Screening by Hybridization
Plaque/colony lifting Making labeled probes Hybridization Pre-hybridization Hybridization Washing Detection Radioactivity Luminescence Florescence Chemiluminescence Rescuing clones and further analysis

Plaque/Colony Lifting
Nylon membrane

Plaque/Colony Lifting

Master plate Replica membrane Labeled nucleic acid probes (radioactive or non-radioactive) Hybridization

32P

Labeled probes hybridize to DNA bound on membrane

Wash off unbound probe Hybridization bottle Place Bio-Max film onto hybridized membrane

Develop film

Hybridization bottle
Original master plate

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

The Result of 2nd Round Screening

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Making Probes
Methods of Detection Radioactive (e.g. P32, P33, S35) Non-radioactive (e.g. biotinylation, horseradish peroxidase, Digoxigenin)

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Example of Non-Radioactive Probes (from Boehringer Mannheim)

DIG (Digoxigenin)

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Example of Non-Radioactive Probes (from Boehringer Mannheim)

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Making Probes
Methods of Synthesis DNA Nick translation (E. coli polymerase I) Random priming (Klenow) Random labeling by PCR 5-end labeling (DNA kinase) 3-end labeling (T4 DNA polymerase, terminal transferase, modified T7 DNA polymerase, Klenow, reverse transcriptase)

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Nick Translation

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Nick Translation

Nicks created by DNaseI

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Nick Translation

Gaps created by E. coli DNA polymerase I (5 to 3 exonuclease activities)

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Nick Translation

Gaps filled by E. coli DNA polymerase I in the presence of dNTPs and labeled deoxynucleotides (5 to 3 polymerase activities, proofreading by 3 to 5 exonuclease activities)

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Nick Translation

Reaction repeats to give uniform labeling

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Random Priming

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Random Priming

Heat denaturing + Random primers (hexamers); + Klenow, dNTPs, and labeled deoxynucleotides

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Making Probes
Methods of Synthesis RNA Random labeling by in vitro transcription (DNA dependent RNA polymerase, e.g. Sp6, T3, T7 RNA polymerase) 3-end labeling (DNA independent RNA polymerase + ATP by polyA tail; RNA ligase)

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Making Riboprobes

Promoter

Gene of Interest

Clone gene of interest under the control of a strong, specific, and inducible promoter (e.g. Sp6, T3, T7)

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Making Riboprobes

Promoter

Gene of Interest

Cut with a restriction enzyme to generate a blunt end or 5 overhang at the 3 end of the gene of interest

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Making Riboprobes

Promoter

Gene of Interest

+ DNA-dependent RNA polymerase corresponding to the promoter used, in the presence of NTPs and labeled nucleotides, to generate randomly labeled cRNA probes

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Hybridization and Washing

Prehybridization for reduction of noise to signal ratio Hybridization usually carried out in solutions of high ionic strength to maximize annealing rate 20-25C below Tm smaller volume the better: fast reassociation and less probes to minimize background, hybridize for the shortest time with minimum probes Washing to remove non-specific binding can control the stringency by altering temperature or [Na+]

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Conditions for Hybridization and Washing

For cloning of a specific gene, use high stringency conditions for hybridization (high temperature, with formamide) and washing (high temperature, low NaCl) For cloning of homologous genes (or members of a gene family), use low stringency conditions for hybridization (low temperature, without formamide) and washing (low temperature, high NaCl)

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Factors Affecting Hybrid Stability

Ionic Strength Tm increases 16.6oC each 10-fold increase in monovalent cations (Na+) between 0.01 to 0.40 M NaCl AT base pairs are less stable than GC base pairs in aqueous solutions containing NaCl Each 1% of formamide reduces the Tm by about 0.6oC for a DNA-DNA hybrid Tm is reduced by 1oC for each 1% of mismatching Negligible effect with probes >500 bp

Base Composition Destabilizing Agents Mismatched base pairs Duplex length

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Tm: Transition Mid-Point; Melting Temperature

Temperature at which 50% of the nucleotides of DNA-DNA, DNARNA or RNA-RNA duplex are hybridized.

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

For Hybrids > 100 Nucleotides

DNA-DNA Tm=81.5oC + 16.6log10[Na+] + 0.41(%G+C) 0.63(% formamide) - (600/l) DNA-RNA Tm=79.8oC + 18.5log10[Na+] + 0.58(%G+C) + 11.8(%G+C)2 - 0.5(% formamide) - (820/l) RNA-RNA Tm=79.8oC + 18.5log10[Na+] + 0.58(%G+C) + 11.8(%G+C)2 - 0.35(% formamide) - (820/l)

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

For Hybrids > 100 Nucleotides

l = the length of the hybrid in base pairs [Na+] between 0.01 M to 0.4 M %G+C between 30% to 75% Stability: RNA-RNA > RNA-DNA > DNA-DNA Tm of a double-stranded DNA decreases by 11.5oC with every 1% decrease in homology pH between 5-9; when outside this range, stability of DNA decreases drastically due to protonation or deprotonation of the bases

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

For Oligos 14-70 nucleotides

Tm = 81.5oC + 16.6(log10[Na+]) + 0.41(%G+C) - (600/N) N=number of nucleotides

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

For Oligos < 18 nucleotides

Tm = (#A+T x 2) + (#G+C x 4)

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Factors Affecting Hybridization Rate

Ionic Strength Base Composition Destabilizing Agents Mismatched base pairs Temperature Viscosity Probe complexity pH Optimal rate at 1.5 M Na+ Little effect 50% formamide: no effect; other concentrations: reduced Each 10% of mismatching reduces rate by a factor of two

Duplex length Directly proportional to duplex length Maximum rate: 20-25oC below Tm for DNA-DNA hybrids, 1015oC below Tm for DNA-RNA hybrids Increase rate of membrane hybridization; 10% dextran sulfate increases rate by factor of ten Repetitive sequences increase the rate Little effect between pH 5.0 to 9.0

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Screening for the Right Clones

By PCR or hybridization if DNA sequence information of the target gene or homologous genes, or the amino acid sequence of the target gene product is available By hybridization if DNA fragments of the target gene or homologous genes are available By functional assays, e.g. Yeast functional complementation Microinjection

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Screening by Yeast Functional Complementation

Saccharomyces cerevisiae is a simple eukaryotic system Relatively easy to grow and perform genetic manipulation Mutants, expression vector, transformation protocol, etc. are available

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Cloning by Yeast Functional Complementation

A yeast mutant which is defective in a function of your interest A library containing cDNAs of the organism of interest is inserted in a yeast vector; cDNAs express under an inducible yeast expression promoter

Yeast expression promoter

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Cloning by Yeast Functional Complementation

Yeast mutant is complemented when the transgene expression is induced

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Designing a Suitable Vector

Yeast expression promoter

E. coli replication origin

Multiple cloning site

Yeast replication origin

E. coli selection marker

Yeast selection marker

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

A Case Study: Cloning of Potassium Channels

A yeast mutant which cannot survive on K+-limiting medium A plant cDNA library constructed in a yeast vector;

Yeast expression promoter

Plant cDNAs express under an inducible yeast expression promoter

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

A Case Study: Cloning of Potassium Channels

Survives in K+-limiting medium when the transgene expression is induced

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

A Case Study: Cloning of Potassium Channels

Mutant Transformant Transgene not induced Only the transformant survives on K+-limiting medium Both survive on K+-rich medium Anderson et al., 1992