Vasant Kumar's document discusses methods for mapping and cloning human disease genes. It describes two types of genome mapping - genetic mapping, which looks at genetic recombination, and physical mapping, which determines the physical distance between DNA sequences. Several techniques for physical mapping are outlined, including restriction mapping, fluorescent in situ hybridization, and sequence-tagged site mapping. The document also explains how to identify the gene responsible for a genetic disease using linkage analysis and positional cloning. Examples discussed include the genes for Huntington's disease, BRCA1/BRCA2-associated breast cancer, and genetic modifiers of Huntington's disease expression.

1. MAPPING AND CLONING OF
HUMAN DISEASE GENES
VASANT KUMAR
M.Sc. BIOTECHNOLOGY

2. INTRODUCTION
Genome mapping is used to identify and record the location of genes and the
distances between genes on a chromosome. Genome mapping provided a
critical starting point for the Human Genome Project.

3. DIFFERENT TYPES OF GENOME MAPPING
• There are two general types of genome mapping called genetic mapping and
physical mapping.
• GENETIC MAPPING looks at how genetic information is shuffled between
chromosomes or between different regions in the same chromosome
during meiosis (a type of cell division). A process called Recombination or
‘crossing over’.
• PHYSICAL MAPPING looks at the physical distance between known DNA
sequences (including genes) by working out the number of base pairs (A-T, C-
G) between them.

4. GENETIC MAPPING
• Alfred Sturtevant created the first genetic map of a chromosome from the
fruit fly (Drosophila melanogaster) in 1913.
• Proposed that the frequency of ‘crossing over’ (recombination) between two
genes could help determine their location on a chromosome.
• By finding out how often various characteristics are inherited together it is
possible to estimate the distance between the genes.

5. Illustration showing a genetic map of the chromosomes from the fruit fly
(Drosophila melanogaster). The names of the genes are shown to the right of
each chromosome. Image credit: Genome Research Limited.

6. DNA MARKERS
SINGLE NUCLEOTIDE POLYMORPHISMS (SNPs):
• Which are positions in a genome where either
of two different nucleotides can occur.
• Some members of the species have one version
of the SNP and some have the other version.
• Usually typed with short oligonucleotide probes
that hybridize to the alternative forms and
hence distinguish which is present.
RESTRICTION FRAGMENT LENGTH POLYMORPHISMS;
• When digested with a restriction endonuclease the
loss of the site is revealed because two fragments
remain joined together.
• Nowadays the presence or absence of the restriction
site is usually determined by PCR.

7. DNA MARKERS
SHORT TANDEM REPEATS (STRS):
• Also called Microsatellites.
• Made up of short repetitive sequences of 1–
13 nucleotides in length.
• The number of repeats present in a particular
STR varies, usually between 5 and 20.
• The number can be determined by carrying
out a PCR using primers that anneal either side
of the STR.
• Examining the size of the resulting product by
agarose or polyacrylamide gel electrophoresis

8. PHYSICAL MAPPING
• Physical mapping gives an estimation of the (physical) distance between specific
known DNA sequences on a chromosome.
• The distance between these known DNA sequences on a chromosome is expressed
as the number of base pairs between them.
• There are a several different techniques used for physical mapping. These include:
• Restriction mapping (fingerprint mapping and optical mapping)
• Fluorescent in situ hybridisation (FISH) mapping
• Sequence tagged site (STS) mapping.

9. RESTRICTION MAPPING
• This uses Specific restriction enzymes to cut an unknown segment of DNA at
short, known base sequences called restriction sites.
• Restriction enzymes always cut DNA at a specific sequence of DNA (restriction
site).
• A Restriction map shows all the locations of that particular restriction site
(GAATTC) throughout the genome.
• There a two specific types of restriction mapping – Optical and Fingerprint.
Image credit: Genome Research Limited.

10. FINGERPRINT MAPPING
• In fingerprint mapping the genome is broken into fragments which are then
copied in bacteria.
• The fingerprint map is constructed by comparing the patterns from all the
fragments of DNA to find areas of similarity.

11. Fingerprint mapping formed the basis to the sequencing of the human,
mouse, zebrafish and pig genomes.

12. .OPTICAL MAPPING
• Optical mapping uses single molecules of DNA that are stretched and held in place
on a slide.
• Restriction enzymes are added to cut the DNA at specific points leaving gaps
behind.
• The fragments are then stained with dye and the gaps are visualised under
a fluorescence microscope.

13. FLUORESCENT in situ HYBRIDISATION (FISH) MAPPING
• This uses fluorescent probes to detect the location of DNA sequences on
chromosomes.
The photograph on the left shows Chromosome 17 from four British peppered moths
with fluorescent probes indicating the physical positions of specific genes. The
illustration on the right shows the relative positions of the genes on the
chromosome. Image credit: Adapted from The American Association for the Advancement of Science

14. SEQUENCE-TAGGED SITE (STS) MAPPING
• This technique maps the positions of short DNA sequences (between 200-500
base pairs in length) that are easily recognizable and only occur once in the
genome.
• To map a set of STSs a collection of overlapping DNA fragments from a single
chromosome or the entire genome is required.

15. IDENTIFICATION OF GENES RESPONSIBLE
FOR HUMAN DISEASES
• A genetic or inherited disease is one that is caused by a defect in a specific
gene, individuals carrying the Defective gene being predisposed toward
developing the disease at some stage of their lives.
Image Credit: T A Brown

16. • There are a number of reasons why identifying the gene responsible for a genetic
disease is important:
• Indication of the Biochemical basis to the disease, enabling therapies to be
designed.
• Identification of the mutation present in a defective gene can be used to devise
a screening programme.
• Carriers can receive counseling regarding the chances of their children
inheriting the disease.
• Identification of the gene is a prerequisite for Gene therapy.
REASONS FOR IDENTIFYING THE GENE RESPONSIBLE FOR A
GENETIC DISEASE

17. HOW TO IDENTIFY A GENE FOR A GENETIC DISEASE?
LOCATING THE APPROXIMATE POSITION OF THE GENE IN THE HUMAN GENOME
Genetic mapping is usually carried out by Linkage Analysis, in which the
Inheritance Pattern for the target gene is compared with the inheritance patterns
for genetic loci whose map positions are already known.
Image Credit: T A Brown

18. INTRODUCTION TO LINKAGE ANALYSIS
• Method that allows mapping of disease genes that are detectable only as
phenotypic traits.
• The entire basis of genetic linkage analysis is that recombination events between
two genetic loci on the same chromosome occur at a rate related to the distance
between them.
• The extent of genetic linkage between loci is measured by the recombination
fraction (θ) between them.
• Two loci are said to be genetically linked when the recombination fraction between
them is less than 0.5.

19. LOD (LOG OF THE ODDS) SCORES
• The LOD score represents the logarithm in base 10 of the odds of linkage of a
trait gene at a recombination fraction θ with a particular marker locus compared
with a recombination fraction of 0.5 between the marker and the trait gene.
• Thus the lod score Z at a given value of θ is:
• LOD score of + 3 or greater (equivalent to greater than 1000:1 odds in favor of
linkage) is considered evidence that two loci are linked.
• Lod scores between -2 and 3 are inconclusive and indicate more data are needed.
• If θ is 0.05 at the highest LOD score, it means that the trait and the marker are 5
centimorgans (cM) apart.

20. DESIGNING AND CONDUCTING PARAMETRIC LINKAGE ANALYSES
• The purpose of linkage analysis is to accrue statistical evidence regarding the
cosegregation of a trait and marker alleles within families.
• Ascertainment of large pedigrees is usually the only way an adequate number
of affected individuals can be obtained to provide the linkage analysis with
sufficient statistical power.
Image Credit: T A Brown

21. • Knowledge about the mode of inheritance should have the greatest influence on
the selection of families.
• Families that include individuals who are inbred with many homozygotes provide
the most powerful sample for recessive traits, while multigenerational.
Image Credit: T A Brown

23. IDENTIFICATION OF DISEASE GENES BY POSITIONAL CLONING
• It is a combination of techniques that
has been extraordinarily successful in
finding the genes that cause many
inherited disorders, including some
that affect the cardiovascular system.
• This approach consists of finding a
DNA marker that cosegregates with
the disorder and then using the tools
of molecular biology to examine
systematically the DNA in the vicinity
of such a marker until the gene is
identified.

24. STEP 1 UNDERSTANDING LINKAGE ANALYSIS: THE TOOLS AND TECHNIQUES
• DNA segments carries a disease gene, the genetic location of the disease can be
found simply by testing many randomly chosen DNA markers until one is found
that happens to segregate at high frequency with the disorder.
• Once such a marker is found, an organized and systematic search for the gene in
the vicinity of this anchor marker can be performed.
• Polymorphic markers, making it possible to track the inheritance pattern of a
specific gene in families and, by extension, in the population at large . Eg: STR.
Image credits: Anil.G menon

25. STEP 2 FINDING A DNA MARKER THAT IS TIGHTLY LINKED TO THE DISORDER
• When the DNA from each member of the Pedigree is analyzed with a sufficiently
large number of randomly chosen polymorphic markers, the strongest linkage is
obtained for a marker that has few or no recombination events between it and the
disease locus.
• A genetic map can thus be constructed by positioning Polymorphic markers in the
region containing the disease gene.
Image credits: Anil.G menon

26. STEP 3 PHYSICAL MAPPING OF THE MINIMAL REGION DEFINED BY THE
FLANKING MARKERS
• The physical distance is determined by a variety of methods such as somatic cell
hybrid mapping and pulsed-field gel electrophoresis, and is used to construct a
high-resolution map of the relatively large region being studied.
• The most commonly used method to generate DNA clones that span this region is a
process known as Chromosome walking.

27. SHORTCUTS TO FINDING THE GENE:
THE ROLE OF CYTOGENETIC TRANSLOCATIONS
• Chromosomal location of a disease gene is often hugely expedited by the
detection of Cytogenetic abnormalities, such as translocations and small
deletions that are sometimes visible in the chromosomes of affected individuals.

28. THE HUNTINGTIN GENE
• Huntington’s disease is a hereditary neurodegenerative disorder caused by an
expansion of a repeating CAG triplet series in the Huntington gene on
chromosome 4, which results in a protein with an abnormally long
polyglutamine sequence.
• Inherited in an Autosomal Dominant fashion, so that each child of an affected
parent has a 50% chance of developing the disease. The gene is located on
Chromosome 4p16.

29. GENETIC MODIFIERS OF HD EXPRESSION
• Significant familial aggregation for the age at onset in HD has been reported.
• Using onset ages adjusted for the size of the HD repeat mutation, pairs of
affected siblings were found to have remarkably similar onset ages independent
of the size of the HD repeat.
• Suggestive evidence for linkage was found at 4p16 (LOD = 1.93) ,6p21–23 (LOD =
2.29), and 6q24–26 (LOD = 2.28), which may be useful for the investigation of
genes that modify age at onset of HD.

30. BRCA1- AND BRCA2-ASSOCIATED BREAST CANCER
• Pathogenic mutations in BRCA1 or BRCA2 are only detected in 25% of families with a
strong history of Breast Cancer, though hereditary factors are expected to be
involved in the remaining families with no recognized mutation.
• Germline mutations in BRCA1 and BRCA2 confer high risk of breast and ovarian
cancers, the penetrance of these genes is incomplete.

31. • The first breakthrough in this project occurred
in 1990 as a result of Restriction fragment
length polymorphism (RFLP) linkage analyses
carried out by a group at the University of
California at Berkeley.
• Study showed that in families with a high
incidence of breast cancer, a significant number
of the women who suffered from the disease
all possessed the same version of an RFLP
called D17S74.
• STR linkage mapping reduced the size of the
BRCA1 region from 20 Mb down to just 600 kb.
PROFILING OF HEREDITARY BREAST CANCER

32. BREAST CANCER REGION
• 100 kb gene, made up of 22 exons and coding for a
1863 amino acid protein, that was a strong
candidate for BRCA1.
• Transcripts of the gene were detectable in breast
and ovary tissues, and homologs were present in
mice, rats, rabbits, sheep, and pigs, but not
chickens.
• Genes from five susceptible families contained
mutations (such as frameshift and nonsense
mutations) likely to lead to a nonfunctioning
protein.
• Gene involved in transcription regulation and DNA
repair, and that both act as tumor suppressor
genes, inhibiting abnormal cell division.

33. REFERENCES
• GENE CLONING AND DNA ANALYSIS AN INTRODUCTION T.A. BROWN, Sixth Edition.
• HOW DO YOU MAP A GENOME? yourgenome.org
• IDENTIFICATION OF DISEASE GENES BY POSITIONAL CLONING Anil G. Menon,
Charles A. Klanke, and Yan Ru Su
• ANALYSIS OF GENETIC LINKAGE, Rita M. Cantor
• HUNTINGTON’S DISEASE GENETICS Department of Neurology, Boston University
School of Medicine, Boston, Massachusetts
• HEREDITARY BREAST CANCER: Clinical, Pathological and Molecular Characteristics
Martin J Larsen, Mads Thomassen, Anne-Marie Gerdes,Torben A Kruse

34. THANK YOU 
A Presentation by VASANT KUMAR