Fragmenting genomic DNA for cloning

– Random methods are best•Mechanical shearing: sonication,

nebulizer•Nuclease treatment (usually restriction

digest): 4 base cutters, partial digest

– Large fragments better than small, fewer clones to get coverage of large genome

Random fragmentation of genomic DNA:

Hydrodynamic shear (physical breakage)-- sonication (vibrating metal probe)-- nebulization (like asthma inhalers)-- passage through small needle orifice

DNA must be repaired with DNA polymerase after these treatments

Enzymatic breakage-- Restriction enzyme (4 cutter, partial

digest)CviJ (pyGCpy and puGCpu)

-- DNAse I (semi-random cleavage)

Partial digest

Size fractionate

Block EcoRI sites

Add linkers

Digest with EcoRI

Ligate to lambda

Package

Early library

construction

Improved library

construction

Partial digest: Sau3A (BamHI compatible ends

Phosphatase

Ligate to lambda

Package

Improved lambdas for libraries• More restriction sites

• Sequences for phage RNA polymerase transcription (useful for probe synthesis)

But….

• Cosmids• BACs• PACs• YACs

…can be used for cloning larger DNAs using similar methods…

Why use lambda libraries?

• Cosmids replicate as high copy number plasmids--tend to be unstable, deleting insert DNA (to reduce drag on cells)

• BAC and YAC libraries difficult to prepare

• larger-sized DNA more difficult to work with

Cloning cDNAs

• Prepared by reverse transcription of mRNA

• Eukaryotic mRNAs--lack introns, often show variable splicing, cDNAs of these RNAs indicate how genes are actually expressed

• Individual mRNA abundance varies widely: to isolate low abundance mRNAs by cDNA cloning, need to make libraries

Key points of cDNA cloning

1) mRNA source (tissue type) matters a lot

2) mRNA must be of high quality (no Rnases….)

3) Rare mRNAs can be enriched

e.g. “Subtractive cloning”hybridize sample cDNA against immobilized RNA/cDNA from a “driver”, clone only those mRNAs that are not bound by the driver

This relies on differential mRNA expression between sample and “driver” mRNA populations

Gubler/Hoffman method (MC Chapter 11)

1) Synthesize first strand cDNA

2) Second strand cDNA

3) Methylate cDNA

4) Attach linkers or adaptors for cloning

5) Fractionate cDNA by size (select 2-8 kb)

6) Ligate cDNA into bacteriophage arms

cDNA libraries

cDNA synthesis

• Make the first DNA strand from the mRNA template using reverse transcriptase

• Remove the RNA

• Make the second DNA strand from the first DNA strand

Primers for “first strand” cDNA synthesis

1) Oligo dT (binds polyA tails)

2) Oligo dT with adaptors (restriction sites)

3) Primers linked to a plasmid

4) Random primers

Random priming

Second strand

synthesis: early

methods

Problem step

Loss of some of the mRNA 5’ end

Second strand synthesis--the Gubler/Hoffman protocol

Homopolymer tailing

But many cDNAs are not full-length--how get only full-length

cDNAs?Utilize the 5’ CAP structure on eukaryotic mRNAs:

cDNA library construction

using reverse

transcriptase cDNA Library

Construction Kit (Clontech)

ESTs: Expressed Sequence Tags

• Full length cDNAs hard to get, difficult to scale up

• But short cDNA sequences are often useful– ID and map specific genes– “High throughput” allows very fast

generation of 200-300 bp sequences, or ESTs

• Millions of ESTs in database• Useful in designing “microarrays” (later)

cDNA libraries: the easy way out

Pre-made cDNA libraries (organisms, tissues, variable conditions

Custom made cDNA libraries (you supply the mRNA)

“kits” for making your own cDNA library

(See Table 11-6 of Molecular Cloning for a directory)

Library construction

1) DNA (entire genome…)a) Fragment the DNAb) Clone in lambda phage vector

2) mRNA (only the expressed genes)a) First strand cDNAb) Second strand cDNAc) Expressed sequence tags (ESTs)

Screening libraries for specific genes

(finding the needle in the haystack)

I. Isolating individual clonesII. Screening by sequence

A. HybridizationB. PCR

III. Screening by protein structure/biological function

IV. Gene identification--diseases

Course reading #29

Overview of strategies for cloning genes

Improved library

construction

Partial digest: Sau3A (BamHI compatible ends)

Phosphatase

Ligate to lambda

Package

You want to clone a gene from the human genome…

So you follow the protocol for

Or…buy a kit/premade library…

Basic “lytic” phage life cycle

Lawn of E. coli

100’s to 1000’s of plaques (individual phage infections)

But…which lambda clone (plaque) has the gene of interest????

How many recombinant DNA molecules are required in a library to get complete coverage of a genome?

N = ln(1-p)

ln(1-f)

p = probability of getting a specific piece of DNA

f = fractional size of clone DNA relative to genome

N = number of clones needed

N = ln(1 - 0.99)

ln[1 - (1.7 x 104 / 3 x 109)]

p = probability of getting a specific piece of DNA = 99%f = fractional size of clone DNA relative to genome = 17000 base pairs (lambda capacity) / 3 x 10 9)

N = number of clones needed = 810,000

N = ln(1 - p)

ln(1 - f)

= 810,000

cDNA cloning: this calculation is harder…

Screen by hybridization

Very fast

Applicable to a large number of clones

Can identify clones that are not full length

But you need to know at least some of the sequence of the gene you are after (more on this later)

1) Known sequences: eg. previously cloned cDNA to locate position in genome (identical match exists in library--stringent hybridization conditions)

2) Probes for non-identical but related sequences: finding a related gene in another species (non-identical match--reduce stringency of hybridization)

3) Probing for a gene from a sequenced protein: eg.

his-phe-pro-phe-met

4) Screen by PCR

Design of nucleic acid probes

make synthetic “mixed probe” (typically 16-mers)

“guessmers”: long, degenerate oligo probes

• 40-60 nts, alternative to short, “mixed probe”• Codon uncertainty mostly ignored

– Most common codon used– Increased length improves specificity

• Inosine substitutions at uncertain positions– Inosine pairs with all 4 bases

• Low stringency hybridizations

“Colony hybridization” for ID of clones(like Southern blotting but without DNA isolation/gel electrophoresis)

Plaque-lift hybridization--using a lambda library

Can do this multiple times (replicate experiments)

Alternative to plating: arrayed libraries

• Individual clones of library spotted onto membranes in high density arrays (tens of thousands of genes)

• Membranes probed as described (a la microarrays)

• Standardizable, centralizable

Using genomic DNA libraries for mapping: Chromosome “walking”

• Prior to sequencing

• It is possible to determine the order of clones in a contiguous sequence (contig)

• Genes whose general location is known (by genetic mapping), but whose function is not known, can be found by starting with the genetic marker clone and “walking” away from it

Chromosome walking: how are individual clones in a genomic library positioned relative to each

other?

The data The genome “assembly”

• Probing can be restricted to one direction with RNA probes generated from clone ends

• Beware of “warping” to another chromosome because of repetitive sequence probes

• Use YAC and BAC libraries to take larger steps

Chromosome walking

Improved lambdas for libraries• More restriction sites

• Sequences for phage RNA polymerase transcription (useful for probe synthesis)

Expression libraries--alternative to hybridization

• Gene product (protein) is made (by E. coli) and detected by variety of methods

• Eukaryotic genes: cDNA library is essential (no introns, gene size small)

Screening:• Immunological• Functional

Immunological screening

Detect antibody with secondary antibody conjugated to reporter enzyme for visualization

The plaque lift: kind of like a Western blot

Functional cloning

• Genetic complementation:– Cloned DNA sequence corrects defect in

host strain• Gain of function

– Cloned DNA confers new function to host

Both of these require cloned DNA to be transcribed, translated into functional protein in host (eukaryotic protein in E. coli could cause problems)

And you need a good assay for expression!

Functional complementation: shaker gene

Shaker-2 mice have defects in the inner ear, poor balance, and deafness

The shaker 2 gene encodes myosin XVMutations in the human homolog can cause deafness

Subtractive cloning– Remove cDNAs that are common to two

sources– Useful for isolation and detections of

differentially expressed rare cDNAs

– Example: differential expression from physiological change

– “driver” DNA - immobilized– “test” cDNA (single stranded): labelled and

then annealed to driver DNA– Remaining DNA has no counterpart in the

driver cells--probe library to locate genes– Or use the remaining DNA to probe a

microarray

Screening libraries for specific genes

(finding the needle in the haystack)

I. Isolating individual clonesII. Screening by sequence

A. HybridizationB. PCR

III. Screening by protein structure/biological function

IV. Gene identification--diseases