Gene Mapping & Cloning
Medical Genetics
Determination of the relative positions
of genes on a DNA molecule (chromosome)
and of the distance,in linkage units or
physical units,between them.
Gene Mapping
1911,red-green color blindness localized to
chromosome X
1968,Duffy Blood Group localized to
chromosome 1
Linkage Analysis
A mathematical procedure that analyzes meiotic
recombination frequencies between pairs of genes
to determine whether two loci are linked and,if so,
how closely.
Linkage Analysis
A mathematical procedure that analyzes meiotic
recombination frequencies between pairs of genes
to determine whether two loci are linked and,if so,
how closely.
Linkage Analysis
A mathematical procedure that analyzes meiotic
recombination frequencies between pairs of genes
to determine whether two loci are linked and,if so,
how closely.
Linkage Analysis
A mathematical procedure that analyzes meiotic
recombination frequencies between pairs of genes
to determine whether two loci are linked and,if so,
how closely.
Recombination fraction
Centimorgan,cM
Genetic Marker
A genetic marker is a tag which allows to
identify a place in a genome (locus),is a ‘tag’
near or within a gene of interest,Genetic
markers are usually highly polymorphic.
Genetic Marker
Blood group; Serum protein; HLA
DNA RFLP
DNA STR & DNA VNTR
Single nucleotide polymorphism,SNP
Linkage Analysis
EcoRⅠ EcoRⅠ EcoRⅠ
3.0 kb 1.7 kb
Probe
Neurofibroma,NF1
RFLP
Ⅰ
Ⅱ
Ⅲ
1 2
1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8
Linkage Analysis
Neurofibroma,NF1
RFLP
Ⅰ
Ⅱ
Ⅲ
1 2
1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8
3.0 kb 1.7 kb
4.7 kb
Linkage Analysis
Neurofibroma,NF1
RFLP
Ⅰ
Ⅱ
Ⅲ
1 2
1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8
4.7 kb
3.0 kb
Linkage Analysis
1955,Morton,logarithm of the odds (Lod)
+3 is considered evidence for linkage
-2 is considered evidence for not linkage
Multipoint Linkage Mapping
Three point crosses
Somatic Hybridization
Cell fusion,The experimental formation
of a single hybrid cell with nuclei and
cytoplasm from different somatic cells.
Sendai virus
Polyethylene Glycol,PEG
Electroporation
Somatic Hybridization
Heterokaryon
Somatic Hybridization
Synkaryon
Somatic Hybridization
H brid cell
Somatic Hybridization
Human & rodent cell hybrid
Over cell divisions,the hybrid cell
looses variable numbers of chromosomes.
For unknown reasons,human chromosomes
are preferentially lost,although it is a
random process with regards to which
human chromosomes are lost.
Somatic Hybridization
HAT Selection
1962,homo HPRT-
1964,mouse TK-
H,hypoxanthine
A,Aminopterin
T,Thymidine
HPRT－
TK－
HAT Selection
HAT Selection
TK－
HPRT－
HAT Selection
TK,chromosome 17
Clone Panel Method
Hybrid
clone
Human chromosome
1 2 3 4 5 6 7 8
A + + + + - - - -
B + + - - + + - -
C + - + - + - + -
Somatic Hybridization
Microcell fusion
Regional assignment
Radiation hybrid
FISH (fluorescence in situ hybridization)
DNA fiber-FISH
Chromosome Painting
CGH (comparative genomic hybridization)
In Site Hybridization
Radiation Hybrid
A hybrid cell containing small fragments
of irradiated human chromosomes.
1975,Goss & Harris
Radiation Hybrid
Radiation Hybrid
Radiation Hybrid
1990,Cox,RH of single chromosome
1994,Walter,RH of whole genome
A hybrid cell containing small fragments
of irradiated human chromosomes.
1975,Goss & Harris
Genebridge 4
radiation dose,3,000 Rads
number of hybrids,93
average fragment size,~10 Mb
Stanford G3
radiation dose,10,000 Rads
number of hybrids,83
average fragment size,~4 Mb
Stanford TNG4 (The Next Generation)
radiation dose,50,000 Rads
number of hybrids,90
average fragment size,~800 kb
Human Whole-Genome Panels
http://www-shgc.stanford.edu/
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
Radiation Hybrid
1 0 0 0 0 1 0 0 0 0 0 0
0 0 0 0 1 0 0 0 0 0 1 0
0 0 0 0 0 0 0 0 1 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
0 1 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
1 0 0 0 1 0 0 0 0 0 0
100001000000000010000010000000001000000000
00000001000000000000000000000010001000000
Conserved Synteny
Synteny
Conservation of synteny
Electronic Clone
http://www.ncbi.nlm.nih.gov
http://genome.ucsc.edu
Gene Cloning
Gene cloning is the process in which one
specific gene that encodes the specific protein of
interest is located and copied (cloned) out of
DNA extracted from an organism.
Functional cloning
Positional cloning
Candidate cloning
Functional Cloning
Information about the function of
an unidentified gene is used to isolate
the gene —— either a gene product or a
functional assay is required.
Positional Cloning
A
T
G
G
T
C
T
C
A
C
T
G
C
A
A
C
C
G
A
T
G
G
T
C
T
C
A
C
T
G
T
A
A
C
C
G
Met
Val
Ser
Leu
Gln
Pro
Met
Val
Ser
Leu
Stop
Candidate Cloning
Positional candidate cloning
GDB or GenBank
Known gene or EST
Disorder
Confirmation
Functional candidate cloning
Candidate Cloning
GDB or GenBankKnown gene
Located disorder
Gene
Confirmation