Is the gene function known

1
Cloning human genes
Is the gene function known?
Y
N
Is it highly expressed in any cell?
N
Y
Is it tissue-specific?
Chromosomal localisation
1 cDNA cloning
Y
N
Build up map with markers, YACS, cosmids, PFGE
2 cDNA cloning after enrichment
Is the phenotype selectable?
Y
3 Transfection and cloning
N
Has the protein been purified?
Y
Look for candidate genes
N
4 (Hybridisation) (Degenerate PCR) Database
searching
Is it a member of a gene family or homologous to
a known gene?
Y
1.1
2
1.2
3
Genetic Code
1.3
HPRT-1
4
Cloning hprt gene by transfection
Mouse hprt- cells
1. Transfect human DNA
Rare primary transfectant
2. Select in HAT
1.4
HPRT-2
5
EST (Expressed Sequence Tag) libraries
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cDNA population
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1. Clone cDNAs into vector. Plate out library
2. Pick clones. Extract DNA. Sequence ends with
vector primers
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Sequences deposited in database
1.5
EST2
6
EST (Expressed Sequence Tag) libraries Clusters
1
10
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2
11
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12
3
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4
13
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5
14
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6
15
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16
7
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17
8
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18
9
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B
C
A
3 Unigene clusters
1.6
EST2