This document discusses various strategies for identifying disease genes, including position-dependent and position-independent approaches. Position-independent strategies include identifying a disease gene through knowledge of the protein product, using an animal model of the disease, or DNA sequence knowledge. Positional cloning involves mapping a disease to a chromosomal region and then identifying candidate genes within that region. Techniques like SSCP analysis and heteroduplex analysis are used to identify mutations in candidate genes. The candidate gene approach starts with a known or suspected protein involved in a disease.

1. Presented BY:
Pradeep Jaswani
M.S.c MHG III semester
Jiwaji University Gwalior
(M.P)

2. Contents
Introduction
Principles & strategies in identifying disease gene
Position independent strategies for identifying disease gene
 Identifying disease gene though knowing the protein product
 Identifying disease gene though an animal model
 Identification of a diseases gene using position independent
DNA sequence knowledge
Positional cloning
 Single stand conformation polymorphism analysis
 Hetero duplex analysis
Candidate gene approach

3. INTRODUCTION
“Identifying genetic determinants of human
phenotypes”.
The approach described are equally applicable
to identifying determinants of diseases or of
normal variations such as red hair or red-
green color blindness.

4. PRINCIPLES & STRATEGIES IN IDENTIFYING
DISEASE GENE
• There are many different ways of arriving at
the final identification, but all path converge
on a candidate gene.
• One way or another, a candidate gene is
identified; the researcher then test the
hypothesis that these gene is disease gene by
screening it for mutation

5. • Candidate genes may be identified without
reference to their chromosomal location but
more commonly, first a candidate chromosomal
region is pinpointed, and then candidate genes
are identified from within that region.
• Positional information reduces the list of possible
candidates from all 30000 or so on human genes
to may be 10-30genes in a candidate region.

6. • Over and over again, when a disease gene is
finally identified, it remains a complete mystery
why mutation should cause that particular
disease
• For e.g. why should loss of function of the
FMR1 protein, involved in transporting RNA
from nucleus to cytoplasm cause, cause mental
retardation (Fragile-X syndrome)

7. POSITION INDEPENDENT STRATEGIES FOR
IDENTIFYING DISEASE GENE
Historically, the first diseases genes were
identified by position independent methods,
simply because no relevant mapping
information existed an no techniques were
available to generate it.
Under those circumstances the candidate must
be suggested by the knowledge of the gene
product: β-globin for sickle cell disease,
phenylalanine hydroxylase for PKU.

8. E1
B1 N D1 Genetic
A1 Y C1 Collect Successfully
Mapped Candidate mapping:
chromosomal families located?
candidate genome
region? for N
homolog? search
mapping
Y D2 E2
C2 Clone the Think again
N B2 Chromosoma chromoso about mode of
Check l deletions or mal inheritance,
A3 heterogeneity
database translocation breakpoints
Cloned s
for genes D3 E3 Think again
candidate Identify new
B3 about
homolog? human candidate
B3 genes gene & go to
Possible A3 B2 D3
D4
candidate Work out full N
gene sequence & E5
Y N structure Pathogenic
B4 mutations
C5 D5
Has it been found?
Collect Look for
fully Y
Y unrelated mutations
Model characterized patients
organism Database searching Laboratory work success
Clinical Input

9. IDENTIFYING DISEASE GENE THOUGH KNOWING THE
PROTEIN PRODUCT
Modern proteomic technique allow even very
tiny quantities of protein to be identified or
partially sequenced by mass
spectrophotometery.
As only one of the nucleotide in the mixture will
corresponding to the authentic sequence, it is
important to keep the number of different
oligonucleotides low so as to increase the
chance of identifying correct target.

10. The number of possible permutations should be
reduced by ligating the target cDNA to a vector
and using one vector-specific primer and one
degenerate protein specific-primer
Host cells containing clones with the desired
gene should produce the protein or at least
parts of the protein, and could be indentified
using colony filters from the library with an
appropriate antibody.
A more rapid alternative is to use partially
degenerate oligonucleotides as PCR primers

11. IDENTIFYING DISEASE GENE THOUGH
AN ANIMAL MODEL
• Many human diseases genes have been
identified with the help of animal models-but
nearly always this has been after checking
positional information's
A mouse mutant and a phenotypically similar
human diseases are mapped to chromosomal
location that are corresponds.
If the mouse gene is cloned its human homolog
became a natural candidate.

12. • Alternatively a diseases gene may be identified
in the mouse and then the human homolog
isolated; this can be mapped by fluorescence
in situ Hybridization, and becomes a candidate
gene for any relevant diseases mapping to that
location.
• This is how the MIFT gene was identified as a
cause of type2 waardenburg syndrome (Hughes
et al 1994).

13. IDENTIFICATION OF A DISEASES GENE USING POSITION
INDEPENDENT DNA SEQUENCE KNOWLEDGE
• Positional independent candidates are also
generated by expression array experiment in
which mRNA samples from patient and
controls are compared to produce a list of
genes whose expression is alter in the disease.
• An interesting application of positional
independent DNA sequence knowledge is
attempt to clone genes containing novel
trinucleotide repeats.

14. • The repeat expansion detection method of
schalling et al 1993 permits detection of
expanded repeats in unfractionated genomic
DNA of affected patient and method have been
developed for cloning expanded repeats
detected (koob et al 1998).

15. Positional cloning
A diseases is identified knowing nothing except its
appropriate chromosomal location.
The first successful application was identification of
the gene for X-linked chronic granulomatous
disease (Royer- Pokora et al 1985)
The successful conclusion of these work in 1986
marked the start of triumphant new era for
human molecular genetics.
One after another, the genes underlying important
disorder such as cystic fibrosis, Huntington's
diseases.

16. Define the candidate region
Obtain clones of all the DNA of the region
Identify all the genes in the region
Prioritize them for mutation screening
Test candidate genes for mutation in affected people
Fig: Logic of positional cloning

17. A NUMBER OF POSITIONAL CLONING METHODS
ARE USED AS FOLLOWS:-
Single strand conformation polymorphism
(SSCP)
Denaturing gradient gel electrophoresis
(DGGE)
Heteroduplex analysis
Chemical mismatch cleavage protein
truncation test (PTT)

18. SINGLE STRAND CONFORMATION POLYMORPHISM
(SSCP)
Reference: http://www.wikilectures.eu/index.php/DNA_Diagnostic_Direct_Methods

19. CANDIDATE GENE APPROACH
• A functional/candidate gene cloning project
starts with either the known protein that is
responsible for an inherited disorder or a
protein that is considered a likely candidate
based on the symptoms and biochemistry of
the disease.
• The amino acid sequence of the protein is
used to deduce the possible cloning sequence
of the corresponding gene.

20. Fig: candidate
gene approach Known (or candidate) protein
Deduce nucleic acid sequence
Examine human genome database
Retrieve bacterial
Localize Characterize gene
artificial
chromosome Identify exons chromosome (BAC)
structure
region clones
Develop Devise DNA
mutation Mutation
diagnostic
detection assays phenotype studies
tests

21. REFERENCES
 HUMAN MOLECULAR GENETICS “Tom strachan &
Read”
 AN INTRODUCTION TO HUMAN MOLECULAR
GENETICS “Jack J.Pasternek”
http://www.wikilectures.eu/index.php/DNA_Di
agnostic_Direct_Methods