Embed Size (px)
Citation preview
Chapter 1Chapter 144. Gene Diagnosis. Gene Diagnosis
Analyzing the origin of extra chromosome 21
？
p1 p2p3p4
Gene diagnosis
To identify the cause of a disease or condition based on gene analysis or test.
Methods of diagnosing a genetic disease:1) Direct diagnosis: detect disease-causing mutation2) Indirect diagnosis: tracking the disease gene by linkage analysis
Direct diagnosis Premise: for the known gene mutations
Point mutation (without size change)– Restriction site altered by mutation
PCR-RFLP– No restriction site altered by mutation
ASO Insertion or deletion (with size change)
– large fragments – by Southern– small fragments – by PCR
Restriction site altered by mutation
• Sickle cell anemia：Hb S
1949 Pauling
ＳＡ
1956 Ingram chain 6th amino acid: glu→val
Loss of the RE MstⅡ site （-CCTNAGG-）
A5’---GTG CAC CTG ACT CCT GAG GAG AAG TCT GCC---3’
glu
S5’---GTG CAC CTG ACT CCT GTG GAG AAG TCT GCC---3’val
Detection1. Southern blot - RFLP
1.15 kbMstⅡ MstⅡ MstⅡ
Normal
1.35 kbMstⅡ MstⅡ
Mutant
(loss of MstⅡ)
2. PCR - RFLP
Southern blot - RFLP
Sickle Cell Disease
(aa)
Heterozygote(Aa)
Normal(AA)
1.35kb
1.15kb
Sickle Cell Disease
(aa)
Heterozygote(Aa)
Normal(AA)
250150100
PCR-RFLP
250150100
AA
DNA Diagnosis of HbS
Aa?
aa
Aa Aa
Detecting KRT9 mutation by PCR-RFLP
GAACTCCTTGAG
5’3’
3’5’
GAATTCCTTAAG
5’3’
3’5’
Allele a
Allele A200bp 100bp
Genomic DNA
300bp productsPCR
EcoR Ⅰ
300bp 200bp100bp
electrophoresis 100200300
Aa AA aa
No restriction site altered by mutation
A5’---GTG CAC CTG ACT CCT GAG GAG AAG TCT GCC---3’
S5’---GTG CAC CTG ACT CCT GTG GAG AAG TCT GCC---3’
ASO
ASO
probeA--TG ACT CCT GAG GAG AAG TC
probeS--TG ACT CCT GTG GAG AAG TC
Spot hybridization
Spot hybridization
For this example, don’t consider the restriction site.
Detecting point mutation by ASO in a PKU family
Probe m
Probe n
aaAa Aa Aa AAAA
Detect size change by Southern blot
5’ 3’16p
5’ 3’16p
-thalassanemia (-globin chains are deficient)
Normal
Silent carrier
Barts hydrops fetalis(fetal or early neonatal death)
or / Minor
Hb H diseaseHb H disease(mild to severe anemia)
10kb
14kbnormal allele
mutant allele
BamHⅠ BamHⅠ
Genomic DNA Bam HI DNA fragmentsElectrophoresis
Size fraction Transfer DNA fragments on membrane Hybridizing
14kb10kb
AA Aa aa
1 2
1 2
Ⅰ
Ⅱ
Southern blot：
Ⅱ1 Ⅰ1 Ⅰ2 Ⅱ2
10kb
14kbⅡ2 is normal.
Duchenne Muscular Dystrophy17 18 19 44 45 46 47 48 49
1719444548
C P1 P2 P3 P4Patient1: exon 44 deleted
Patient2: exon 17 deleted
Patient4: exon 19 deleted
Patient3: exon 45 deleted
Detect size change by PCR
1 2
1 2
Ⅰ
Ⅱ
A DMD family
normal
Indirect diagnosis
Demonstration of a mutation in known gene
Demonstration of a disease allele of unknown genes by using tightly linked markers to the disease gene
Indirect Gene Diagnosis
M1 1 23 2 2 4 4 12 2
XAY XAXa
XaY
XaY XAY XaY
? ?
Disease gene
Marker 1
Recombination ?
Detect a mutation in a known gene
Hemophilia A
1 1/2
2 1/1 2 1/2 2 1
XAXa
XaY XaY
XAY
XAY XAYXAXA XAXa
Ⅰ
Ⅱ1
2
2
1
1
3 4 5 6
Ⅲ 2/2
1/2
1
2
XAY
XaY
XAXA
XAXa
Disease gene
Marker 1 Marker 2
E.g. BDB; AD; tightly linked to D9S938,which is STR; there are 7 alleles in normal population.
2/5
患
Ⅰ
Ⅱ1
2
2
1
1
3 4 5 6
Ⅲ
2/4 3/5
2/3 4/5 2/5 3/4 4/52/5
2
2/4
患
3/5
常
3/4
常
2/2 患
2/5 ？
5/5 常
Detect a mutation in an unknown gene (mapped disease gene)
Aa aa
Aa Aa aa aa
Premise Family data Tightly linked polymorphic marker Heterozygote Location known
AD-delayed. According to the individual genotypes of the polymorphic
marker tightly linked to the mutant gene, write out the genotypes of the false gene.
Ⅰ
Ⅱ
Ⅲ1
1
1 2
2
2 3
3 4
4
5 6
3/5 2/6
5/6 2/5 2/5 2/3 1/61/4
1/5 1/6 2/5 1/2
Example
A is linked to 5:
Ⅰ
Ⅱ
Ⅲ1
1
1 2
2
2 3
3 4
4
5 6
3/5 2/6
5/6 2/5 2/5 2/3 1/61/4
1/5 1/6 2/5 1/2
Aa
Aa Aa
Aa
aa
aa aa
aaaa
?
