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Isolation of genes controlling traits of interest
The biggest limitation to recombinant DNA approaches to plant
and animal improvement is that we have not identified the genes
we need to manipulate.
I
Methods that require no knowledge of the gene product
•Positional (map based) cloning
•Insertional inactivation
•Complementation (transformation of whole library)
II
Methods that use information on the gene product
•Homology based
•Based on expression pattern
Requirements for Positional Cloning
•DNA markers (RFLPs, PCR markers etc.) that are
genetically tightly linked to the gene.
• Large family segregating for the gene and the
DNA markers.
•Easiest for trait controlled by single gene,
preferably an easy to classify phenotype.
Review of genetic linkage
Linkage between genes can be defined by three examples
1. Independent assortment (unlinked)
AaBb X aabb
Parental genotypes
0.25 AB
0.25 Ab
0.25 aB
0.25 ab
X All ab
0.25 AaBb
0.25 Aabb
0.25 aaBb
0.25 aabb
X All ab
0.50 AB/ab
0.50 ab/ab
}
New
combinations
2. Complete linkage
AB/ab X ab/ab
Parental genotypes
0.50 AB
0.50 ab
No new
combinations
3. Incomplete linkage
AB/ab X ab/ab
Parental genotypes
0.4 AB
0.1 Ab
0.1 aB
0.4 ab
}
0.4 ab
0.1 ab
RX
0.1 ab
0.4 ab
}
R
0.4 AB/ab
0.1 Ab/ab
0.1 aB/ab
0.4 ab/ab
}
New genetic
recombinations
Review of genetic linkage
Linkage occurs when two genes are near each other on the same
chromosome. Their ‘linkage distance’ can be determined by seeing
how frequently they segregate together. Consider two linked genes
in a heterozygous individual:
A
B
a
b
The gametes from this individual will be AB or ab unless a crossover
occurred between them during meiosis.
A
B
a
X
b
Crossovers produce Ab and aB gametes. The % of these
recombinant gametes is essentially the linkage distance. For
example, 40 AB, 40 ab, 10 aB and 10 Ab progeny gametes would be 20
% recombination or roughly 20 map units.
Crosses for estimating map distances
The main thing that complicates linkage analysis, is that the progeny
from many types of crosses, can not be definitely classified as being
from recombinant gametes! Especially, in when gametes from both
parents are segregating. Analysis is simple when crossing haploids,
Parents
Transient diploid
AB x ab
AB
ab
Progeny
AB
ab
Ab
aB
or when only the gametes of one diploid parent are segregating;
Parents
AB
ab
Progeny
X ab
ab
AB
ab
ab
ab
Ab
ab
aB
ab
In more complicated crosses, or if analyzing many genes, you
might use a computer program to calculate linkage.
Visualization of Restriction Fragment Length Polymorphism (RFLP)
Probe
Same chromosome segment A
B
in 4 different genotypes
DNA is cut with EcoRI and
Run on Gel
A B C D
C
D
E
E
E
E
E
E
E
E
E
E
A B C D
_
DNA in gel is made singlestranded, Transferred to a
membrane, and probed with
s.s 32P-labeled probe.
Membrane (blot) is exposed
to X-ray film to see
polymorphic bands
+
E
RFLPs, continued…….
RFLPs were the first type of DNA ‘marker’. ‘Loci’ (places on
chromosomes) are sometimes called markers because they may not
represent actual genes, but represent a locus, just like a gene does.
R M1
R is your favorite gene. Like all genes it
maps to a specific chromosomal
position.
Marker 1 is an RFLP marker. The probe used to
identify the bands on the gel-blot is a DNA
sequence (maybe a gene) that occurs at this
position and no where else. Marker 1 is linked to
R.
Calculate a map distance between a dominant disease resistance
gene ‘R’ and a marker gene M
Cross:
R M X
R M
r m
r m
R M
r m
r m
r m
X
30 Progeny:
R,M/r,m
r,m/r,m
R,M/r,m
R,M/r,m
r,M/r,m
r,m/r,m
R,m/r,m
R,M/r,m
r,m/r,m
r,m/r,m
What is your estimate of map distance?
R,M/r,m
R,M/r,m
r,M/r,m
r,m/r,m
r,m/r,m
r,m/r,m
R,M/r,m
r,m/r,m
R,M/r,m
R,M/r,m
r,m/r,m
R,M/r,m
R,M/r,m
R,M/r,m
R,m/r,m
R,M/r,m
r,m/r,m
R,M/r,m
r,m/r,m
R,m/r,m
The same cross with Phenotypes given, instead of Genotypes.
Parents
S R F1
Progeny: F1 X susceptible parent
M
m
S R R
Phenotype
S R S R S S R S S R S R S R R S R R S R R
Molecular markers (M or m)
(R = Resistant, S = Susceptible)
What is your estimate of map distance?
Positional cloning, continued…….
If your marker is close enough to R, you might be able find a
large clone (e.g. BAC) that contains both the gene and your
marker sequences.
R M1
Arabidopsis has an average of about 26,000 Kb on each
chromosome (N=5), or about 13,000 Kb/chromosome arm. This
is the equivalent of 108 BAC clones averaging 120 Kb each, laid
end to end. Other plants and mammals generally have much
larger chromosomes.
Your marker has to be very close to your gene to be on the same
clone!
Positional cloning, continued…….
If you are fortunate enough the marker is within range of a single
BAC clone of your gene….Now how do you proceed?
R M1
BACs
R
M1
R
M1
1. Sequence the BAC
2. Use complementation to prove you have the gene
3. Mutagenize then compare mutants to normal gene
…However, in most cases researchers are not that lucky.
Positional cloning, continued…….
If your marker is not within range of a single BAC clone of your
gene, you may be able to arrange a series of overlapping BAC
clones that will span the distance.
R M1
R
M1
BAC
Contig
A series of overlapping clones, contiguous with a segment of
chromosome, is called a contig.
Genetic recombinants are used to orient clones/contigs Example:
Chromosome segment in parents for mapping population.
Parent 1
Parent 2
M1
R
M2
m1
r
m2
Chromosome segments in sample of nonrecombinant progeny:
1
2
M1
R
M2
m1
r
m2
Selected progeny derived from crossovers between markers 1 and 2:
51
151
261
321
411
m1
R
M2
m1
r
M2
m1
R
M2
M1
r
m2
m1
r
M2
Contig of cloned fragments, isolated using M1 and M2:
M1
M3
M2
Genetic recombinants are used to orient clones/contigs, continued...
Selected progeny derived from crossovers between markers 1 and 2:
51
151
261
321
411
m1
R
M2
m1
r
M2
m1
R
M2
M1
r
m2
m1
r
M2
M3
Standard RFLP analysis
using the recombinants
Probe/
Allele
M1
m1
P1 P2
(R) (r)
1 2
(R) (r)
51 151 261 321 411
(R) (r) (R) (r) (r)
*
m2
M2
M3
m3
Conclusion, M3 is closer
* *
*
*
Genome size effects feasibility of positional cloning
Genome size
Yeast
Neurospora
Drosophila
Arabidopsis
Rice
Sorghum
Tomato950
Human
Maize
Barley
Physical (Mb)
15
50
150
130
430
750
1500
3400
2500
5000
Kb/cM*
3
30
300
130
330
400
Recombinational (cM)
5000
1000-1500
500
1000
2100
1800
650
3000
1800
1500
1000
1400
3000
*Kb/cM can be a very rough guide to how physically far a linked marker is
from a gene
If you printed the human genome in a 12 character/inch font, how far would it go?
Arabidopsis
Human
Wheat
How do you find your gene after you have it narrowed
down to a specific cloned interval?
•Complementation? (transform your clones into ‘mutant’
line to ‘complement’ the phenotype)
•Sequencing the whole clone(s)?
How do you verify that it is the gene you are looking for?
•Complementation by transformation?
•Analysis of multiple mutants?
DNA FINGERPRINTING
RFLP- Restriction Fragment Length Polymorphism
PCR based fingerprinting
AFLP- Amplified Fragment Length Polymorphism
SSR- Simple-Sequence-Repeats
(microsatellites)
STR-short tandem repeats
RAPD- random amplified polymorphic DNA
SNP- single nucleotide polymorphisms
}
PCR: Amplifying DNA with a heat-stable polymerase
Reaction mix: Template DNA, 4 nucleotides, primers, buffer, polymerase
Step 1) Melt template DNA (~96o C)
Step 2) Let primers anneal to template DNA by dropping temperature.
5’
3’
3’
5’
Step 3) Extend new strands at ~68oC
5’
3’
5’
3’
5’
3’
3’
5’
PCR: Amplifying DNA continued
Step 4) Repeat melting, annealing and extension steps
5’
3’
3’
5’
5’
3’
3’
5’
Step 4 - 30) Re-repeat melting, annealing and extension steps
DNA between the primers
is amplified many times.
DNA polymerase reaction
Denature
Strand
primers
dNTPs
polymerase
synthesis
DNA Polymerases: several different enzymes from different sources
and with different properties (Taq polymerases, DNA polymerase I, Klenow)
(optimum temp, heat stability, exonuclease activity)
PCR- polymerase chain reaction
Taq polymerase
Thermus aquaticus
Enzyme active at high temp
(68º to 74º C)
Heat stable
Cycles molecules
1
2
2
4
3
8
30
1 X 109
DNA FINGERPRINTING
RFLP- Restriction Fragment Length Polymorphism
PCR based fingerprinting
AFLP- Amplified Fragment Length Polymorphism
SSR- Simple-Sequence-Repeats
(microsatellites)
STR-short tandem repeats
RAPD- random amplified polymorphic DNA
SNP- single nucleotide polymorphisms
}
How can polymorphism be observed using PCR?
||||||||||||
Line A
||||||||||||
Fragment size determined by
distance between primer sites
||||||||||||
Line B
Deletion changing fragment size
||||||||||||
| || || ||
Line C
||||||||||||
Alteration in primer site
A
PCR amplify DNA from
each line using the two
specific primers, then run
the products on a gel.
B
C
AFLP analysis gives lots of bands on a single gel.
(Amplified Fragment Length Polymorphism)
1) Cut DNA with EcoRI (6 bp recognition) and MseI (4 bp).
E
M
2) Ligate adaptors on to ends
M
E
3) PCR amplify with primers from adaptors. Too many fragments will
amplify to resolve on a gel at this stage.
M
E
4) Perform additional PCR cycles with primers that have additional
selective bases to reduce the number of fragments that amplify. The
EcoRI primer that is 32P labeled.
P
E
M
5) Run the reactions on a long polyacrylamide gel, and expose the gel to
X ray film. Only the fragments with an EcoRI end will expose the film.
AFLP analysis of two nearlyidentical rice lines with and
without a disease resistance
gene.
Enlargement
Gel by Brad Porter & Frank White
Simple-Sequence-Repeats show frequent size polymorphism
Individual A
||||||||||||
ATATATATATATATATATATAT
TATATATATATATATATATATA
Individual B
||||||||||||
ATATATATATATATATATATATATAT
TATATATATATATATATATATATATA
Individual C
||||||||||||
||||||||||||
ATATATATATATATATATATATATATATATATATA
TATATATATATATATATATATATATATATATATAT
||||||||||||
||||||||||||
•If the primers sequences only occur once in the genome, these mark single
loci.
•Because of their frequent size polymorphism, and multiple alleles, these are
very useful markers. Can be used in forensic science.
•They are also called STR or ‘microsatellite’ markers.
Simple Sequence Repeat (SSR)- a small segment of DNA, usually 2 to
5 bp in length that repeats itself a number of times. Useful SSRs usually
repeat the core motif 9-30 times.
Single Nucleotide Polymorphisms (SNPs): a
single base variation between two otherwise
identical DNA sequences.
Random Amplification of Polymorphic DNA
RAPD reactions are PCR reactions, but they amplify segments of DNA
which are essentially unknown to the scientist (random)
Standard PCR:
RAPD detection cont.’
A
B
Cloning by Insertion Mutagenesis
General strategy:
1) Construct a large population (1000’s) that has lots of insertion
mutations from an active transposon or from transgene insertion
2) Find a individual that has the expected phenotype of a mutant of YFG
3) Use the cloned transgene/transposon as a probe to clone the
mutated gene out of a library of clones made from the mutant
4) Use the cloned mutant gene to isolate the wild-type gene from a
library of a non-mutant individual
Example, a resistance gene cloning project
Resistance gene
Transposon
Resistant Parent
Find susceptible mutant
Make and screen library
Cloning by Insertion mutagenesis,
Requirements:
•Easy to score, reliable phenotype, preferably meiotically stable.
•Efficient insertion mutagenesis agent, like;
Highly active, cloned T.E. system (native or introduced)
OR
Highly efficient transformation system, preferably a species
with a high gene density (e.g. fungi, Arabidopsis, rice).
Low gene density, e.g. maize, wheat
High gene density, e.g. Arabidopsis
Cloning by Complementation
Strain 1: Carries gene
Make library of big clones
Genomic fragments
Strain 2: Doesn’t carry gene, or
carries mutant form.
Gene
Vector
•Transform strain 2 with whole strain 1 library.
•Isolate transformed individuals, each carrying a different cosmid.
•Test each individual for phenotype expected for the gene.
•Individual expressing phenotype should have the gene on its cosmid.
Problems:
Need very efficient transformation
Genomes size is a limitation
O.K. for bacteria, 5000 kb genome = ~150 Cosmids
Yeast, 15,000 kb = ~450 Cosmids
Cloning by Homology
Methods:
1) Probe library with heterologous gene (e.g. from another species).
•
Usually have to alter hybridization conditions (reduce stringency).
•
Homology is related to genetic distance and gene conservation.
•
Within taxonomic family usually works.
•
If stringency conditions are too low, hybridization could be bogus.
Perfect homology
ACTCTAGTACTGATCGTCTGATCTA
|||||||||||||||||||||||||
TGAGATCATGACTAGCAGACTAGAT
Less homologous
ACTCTAGTACTGATCGTCTGATCTA
|||| ||| |||| |||||| |||
TGAGCTCAAGACTGACAGACTCGAT
2) Identify the most highly conserved sequences in the gene by comparing
versions of the gene from other species. Then design PCR primers and try
to amplify the gene from your species.
Cloning strategies based on gene expression patterns.
•The content of cDNA libraries vary with tissue, developmental
stage or environmental conditions.
•Some genes may be very abundant in some libraries, e.g. seed
storage proteins in endosperm libraries.
•Others libraries may have gene in lower abundance, but they
are specific to that library. Its possible to find the specific genes
by ‘subtracting’ the sequences present in other tissues. E.g. A
library from pathogen-infected tissue with the sequences from
uninfected tissue subtracted out.
•Expression patterns of a gene may make it a ‘candidate’ for
genes controlling a phenotype.