Transcript Fragmenting genomic DNA for cloning

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….
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•
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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 DNA
b) Clone in lambda phage vector
2) mRNA (only the expressed genes)
a) First strand cDNA
b) Second strand cDNA
c) Expressed sequence tags (ESTs)
Screening libraries for specific genes
(finding the needle in the haystack)
I.
II.
Isolating individual clones
Screening by sequence
A.
B.
III.
IV.
Hybridization
PCR
Screening by protein
structure/biological function
Gene identification--diseases
Course reading #29
Overview of strategies for cloning genes
You want to clone
a gene from the
human genome…
Improved
library
construction
Partial digest:
Sau3A (BamHI
compatible ends)
So you follow the protocol for
Phosphatase
Ligate to lambda
Package
Or…buy a kit/premade library…
Basic “lytic” phage life cycle
100’s to 1000’s of
plaques (individual
phage infections)
Lawn of E. coli
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 - p)
ln(1 - f)
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
ln(1 - 0.99)
N=
ln[1 - (1.7 x 104 / 3 x 109)]
cDNA cloning: this calculation is harder…
= 810,000
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)
Design of nucleic acid probes
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
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”
Chromosome walking
• 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
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
The plaque lift: kind of
like a Western blot
Detect antibody with secondary antibody conjugated
to reporter enzyme for visualization
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
The shaker 2 gene encodes
myosin XV
Mutations in the human
homolog can cause deafness
Shaker-2 mice
have defects in
the inner ear,
poor balance,
and 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.
II.
Isolating individual clones
Screening by sequence
A.
B.
III.
IV.
Hybridization
PCR
Screening by protein
structure/biological function
Gene identification--diseases