Mapping a gene defined by the mutation Lecture 4, 5, and 6Lecture 4, 5, and 6: Read chapter 7,...
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Mapping a gene defined by the mutation A. Classical genetics B. Molecular mapping I. Molecular techniques: Restriction mapping Library construction and screen PCR✁ (polymerase chain reaction) DNA sequencing (lecture 7) Southern blot II. Molecular markers: RFLP, CAPS, SSLP, SNP III, Molecular mapping C. Map-based cloning Lecture 4, 5, and 6: Read chapter 7, p320-322, 326-327 (box 7.2), 329-330 Fig. 7.17; 7.18, 7.29, 7.30
Transcript of Mapping a gene defined by the mutation Lecture 4, 5, and 6Lecture 4, 5, and 6: Read chapter 7,...
Mapping a gene defined by the mutation
A. Classical genetics
B. Molecular mapping
I. Molecular techniques: Restriction mapping
Library construction and screen
PCR! (polymerase chain reaction)
DNA sequencing (lecture 7)
Southern blot
II. Molecular markers: RFLP, CAPS, SSLP, SNP
III, Molecular mapping
C. Map-based cloning
Lecture 4, 5, and 6:
Read chapter 7, p320-322, 326-327 (box 7.2), 329-330Fig. 7.17; 7.18, 7.29, 7.30
XA B a b
A B a b
A B
a b
A B
a b
A b
a B
Ab + aB
total= rec, freq.
Xa b
a b
90901010
10 + 10200
= 0.1 = 10%
Library construction and screen (Chapter 7, box 7.2)
animation
animation
Mapping a gene defined by the mutation
A. Classical genetics
B. Molecular mapping
I. Molecular techniques: Restriction mapping
Library construction and screen
PCR! (polymerase chain reaction)
DNA sequencing (lecture 7)
Southern blot
II. Molecular markers: RFLP, CAPS, SSLP, SNP
III, Molecular mapping
C. Map-based cloning
Lecture 4, 5, and 6:
Read chapter 7, p320-322, 326-327 (box 7.2), 329-330Fig. 7.17; 7.18, 7.29, 7.30
Ler
Col
HindIII HindIII HindIII
HindIII HindIII
2 kb 5.7 kb
Ler Col Het1kb
ladder
5
2
1
Probe:
RFLP: Restriction Fragment Length Polymorphism
HindIII digestionElectrophoresisSouthern blottingHybridization with the probeautoradiography
Example of RFLP
Restriction digestionGEEthedium bromide stain
BlottingHybridizationX-ray radiography
CAPS (Cleaved Amplified Polymorphic Sequences)
Ler
Col
HindIII HindIII HindIII
HindIII HindIII
2 kb 5.7 kb
Ler Col Het1kb
ladder
5
2
1
PCRHindIII digestionGE
L C M
http://www.arabidopsis.org/index.jsp
Mapping with a CAPS marker
Xf E F e
f E F e
f E
F e
0
1
2
11
6
0 17 X 2 = 0.176
L C M
E/e: CAPS markerF/f: a mutation causing abnormal flowersLer: solid lineCol: dotted line
self
No. of plantsNo. of
recombinant Chr.
Mapping with a SSLP marker
http://www.arabidopsis.org/index.jsp
(AT)20
150 bp
(AT)15
140 bp
Col
Ler
PCR and GE
Ler Col HetM
L C 1 2 3
SNPs (Single Nucleotide Polymorphisms)
http://www.arabidopsis.org/index.jsp
Mapping a gene defined by the mutation
A. Classical genetics
B. Molecular mapping
I. Molecular techniques: Restriction mapping
Library construction and screen
PCR! (polymerase chain reaction)
DNA sequencing (lecture 7)
Southern blot
II. Molecular markers: RFLP, CAPS, SSLP, SNP
III, Molecular mapping
C. Map-based cloning
Lecture 4, 5, and 6:
Read chapter 7, p320-322, 326-327 (box 7.2), 329-330Fig. 7.17; 7.18, 7.29, 7.30
Yeast Artificial Chromosomes YAC (Fig. 7.30)
YAC: 100-1000kb yeastBAC: 80-300kb bacteriumCosmid: 20-50 kb bacteriumLamda: 10-20kb bacteriumPlasmid: 0.2-15kb bacterium
Vector insert size Host
Physical map (Fig. 7.29)
In Arabidopsis: 1% recombination = about 200 kb
0.1% = 20kb
Mapping with a CAPS marker
Xf E F e
f E F e
f L
F C
0
1
2
11
6
0 17 X 2 = 0.176
L C M
E/e: CAPS markerF/f: a mutation causing abnormal flowersLer: solid lineCol: dotted line
self
No. of plantsNo. of
recombinant Chr.
f E
0 1 1 1 1 1 1 (6/34) = 17% (3500kb)
cs3 cs2
The larger the mapping population
The more recombinants between two markers
The higher the resolution of the map
The closer the gene f is from the nearest molecular marker
(i.e. 1/600 kb)
cs7 cs6 cs5 cs4 cs3 cs2
(60/340)
(1/60 kb)
1 1 1 1 1 1 1 1 1 1
lsnlsn-1-1 is flower mutant is flower mutant
Wild type lsn-1
CTR1
1K 14K 30K 60K87K
1/176 0/176 1/176
6 5 4 3 5 6 9
T10O8PDC3 T22P11F15A17F7J8 T20L15
2853326767 29885
Cla1BamH1 Cla1
WT: TACCCGAALsn-1: TACTCGAA
T7H20
Map-based cloning of LSN
Create F2 mapping population
establish linkage
identify nearby PCR markers
Assay a large number(1,000) of F2 plants with flanking markers
Establish physical map& contig
More fine mapping
Transformation rescue (complementation)and/or
sequence mutant alleles
Identify the gene
Steps in map-based cloning
Download Problem set #1 from Bsci 411 websiteDue next Tuesday (2/24/04)
