Gene mapping involves determining the physical location of genes on chromosomes. There are two main types of gene mapping: genetic mapping and physical mapping. Genetic mapping uses genetic techniques like linkage analysis to construct maps showing relative gene positions based on recombination frequencies. Physical mapping uses molecular biology techniques to directly examine DNA and determine absolute positions of genes and sequences. Key methods in physical mapping include restriction mapping, fluorescence in situ hybridization (FISH), and sequence tagged site (STS) mapping. Gene mapping is important for understanding genetic diseases and developing gene therapy methods.

1. Gene Mapping Methods
Presented by-
Meenakshi Das
MSc., PGDBI

2. Introduction
• Gene mapping means the mapping of genes
to specific locations on chromosomes.
• It is very important in the understanding of
genetic diseases.
• Such maps indicates the positions of genes in
the genome and also distance between them.

3. History
• In 1911, by Thomas Hunt Morgan, gene for eye-
color was located on the X chromosome of fruit
fly. (1)
• E.B. Wilson attributed the sex-linked genes
responsible for color-blindness and hemophilia in
human beings to be located on the X-
chromosome, similar to the many X-linked factors
being described by the Morgan group in flies.

4. Types Of Gene Mapping
• Genetic mapping
– Based on the use of genetic techniques to construct maps.
– These maps show the positions of genes and other sequence features
on a genome.
– Also helps to determine the relative position between two genes on a
chromosome.
• Physical mapping
– Uses molecular biology techniques to examine DNA molecules directly.
– Based on these techniques map construction is done.
– These maps show the positions of sequence features , including genes.

5. Genetic Mapping
• A genetic map must show the positions of
distinctive features.
• Requires informative markers – polymorphic
and a population with known relationships
• Best if measured between “close” markers.
• Unit of distance in genetic maps =
centiMorgans, cM
• 1 cM = 1% chance of recombination between
markers

6. Genes-The first marker to be used
• First genetic map was constructed in early 20th century.
• Genes were the first markers used.
• The first friut fly map showed the positions of genes. For
example- body colour, eye colour etc.
• All these maps were based on phenotype of the organism.
• But visual phenotypes were limited and even a single
phenotype could be affected by more than one genes.
• Therefore more comprehensive and less complex
characteristics were required.

7. Use of Biochemical Markers
• For microbes and humans, biochemical
phenotypes were preferred.
• A big advantage is that relevant genes have
multiple alleles.
• For example- Gene called HLA-DRB1 has at
least 290 alleles and HLA-B has over 400
alleles.

8. DNA Markers for Genetic Mapping
• Genes are useful markers but not ideal.
• Mapped feature that are not genes are called DNA markers.
• DNA markers must have at least two alleles to be useful.
• DNA sequence features that satisfy this requirement are-
– Restriction Fragment Length Polymorphism (RFLP)
– Simple Sequence Length Polymorphism (SSLP)
– Single Nucleotide Polymorphism (SNP)

9. RFLP
• RFLP is the first type of DNA marker to be studied.
• Restriction enzymes cut DNA at specific
recognition sequences.
• But restriction sites in genomic DNA are
polymorphic and exists as two alleles.
• The RFLP and its position in the genome map can
be worked out following the inheritance of its
alleles.

10. RFLP

11. Scoring an RFLP
• There are two methods for scoring an RFLP
– Southern hybridization
– PCR

12. Southern Hybridization

13. PCR

14. Simple Sequence Length Polymorphism
(SSLP)
• SSLPs are arrays of repeat
sequences that display length
variation.
• Here different alleles contain
different number of repeat
sequences.
• SSLPs can be multiallelic.
• Two types of SSLPs are-
– Minisatellites (VNTRs)
– Microsatellites (STRs)
• Microsatellites are more
popular than minisatellites as
DNA markers.

15. Typing of SSLP (STRs) by PCR

16. Single Nucleotide Polymorphism (SNP)
• There are some positions in the genome where some
individuals have one nucleotide while others have another.
• Some SNPs give rise to RFLPs but many do not.
• SNPs originate when a point mutation occurs in a genome
converting one nucleotide to another.
• There are just two alleles-the original sequence and the
mutated version.
• SNPs enable very detailed genome maps to be
constructed.

17. SNP

18. Typing method of SNPs
• These are mainly based on oliginucleotide
hybridization analysis. These are-
– DNA Chip Technology
– Solution Hybridization
– Oligonucleotide Ligation Assay
– Amplification Refractory Mutation Assay (ARMS Test)

19. DNA Chip Technology
Image Source: Internet

20. Solution Hybridization Technology

21. Oligonucleotide Ligation Assay
Image Source:
GENOMES 3

22. Amplification Refractory Mutation
Assay (ARMs Test)
Image Source:
GENOMES 3

23. Linkage analysis is the basis of genetic mapping
• Chromosomes are inherited as intact units, so it was reasoned that the alleles of
some pairs of genes will be inherited together because they are on the same
chromosome. This is the principle of genetic linkage,
• Pairs of genes were either inherited independently, as expected for genes in
different chromosomes, or, if they showed linkage, then it was only partial linkage:
sometimes they were inherited together and sometimes they were not
• The frequency with which the genes are unlinked by crossovers will be directly
proportional to how far apart they are on their chromosome. The recombination
frequencyis therefore a measure of the distance between two genes
• If you work out the recombination frequencies for different pairs of genes, you can
construct a map of their relative positions on the chromosome

24. The LOD Score
• The LOD score often used for linkage analysis in human
populations, and also in animal and plant populations.
Computerized LOD score analysis is a simple way to analyze
complex family pedigrees in order to determine the linkage
between Mendelian traits (or between a trait and a marker,
or two markers).
• The method briefly, works as follows:
– Establish a pedigree
– Make a number of estimates of recombination frequency
– Calculate a LOD score for each estimate
– The estimate with the highest LOD score will be considered the
best estimate

25. LOD Score
• The LOD score is calculated as follows:
• LOD = Z = Log10 probability of birth sequence with a given linkage
probability of birth sequence with no linkage
• By convention, a LOD score greater than 3.0 is
considered evidence for linkage.
• On the other hand, a LOD score less than -2.0 is
considered evidence to exclude linkage.

26. Genetic Mapping In Bacteria
• Transformation
• Conjugation
• Transduction

27. Demerits of Genetic mapping
techniques
• A map generated by genetic techniques is not
much sufficient for directing the sequencing
phase of a genome project. This is for two
reasons:
• The resolution of a genetic map depends on
the number of crossovers that have been
scored.
• Genetic maps have limited accuracy.

28. Physical Mapping
• The most important techniques used in
physical mapping are as follows:
– Restriction Mapping
– Fluorescent in situ Hybridization (FISH)
– Sequence Tagged Site (STS) Mapping

29. Restriction Mapping
• Restriction mapping locates the relative positions
on a DNA molecule of the recognition sequences
for restriction endonucleases.
• The simplest way to construct a restriction map is
to compare the fragment sizes produced when a
DNA molecule is digested with two different
restriction enzymes that recognize different
target sequences.

30. Restriction Mapping
Image Source: GENOMES 3

31. Limitations of Restriction Mapping
• Restriction maps are easy to generate if there are relatively few cut sites for the
enzymes being used.
• Restriction mapping is more applicable to small rather than large molecules.
• In practice, if a DNA molecule is less than 50 kb in length it is usually possible to
construct an unambiguous restriction map for a selection of enzymes with six-
nucleotide recognition sequences.
• This problem can be solved to some extent by choosing enzymes which are
expected to have infrequent cut sites in the target DNA molecule.
• These “Rare cutters” fall in two categories:
– Enzymes with seven-or eight-nucleotide recognition sequences
– Enzymes whose recognition sequences contain motifs that are rare in the target DNA

32. Direct examination of DNA molecules
for Restriction sites
• Two ways of doing this:
– Gel stretching
– Molecular Combing

33. Gel Stretching
Image Source:

34. Molecular Combing
Image
Source:
GENOMES 3

35. Fluorescent in situ hybridization (FISH)
• FISH enables the position of a marker on a
chromosome or extended DNA molecule to be
directly visualized.
• In optical mapping the marker is a restriction site
and it is visualized as a gap in an extended DNA
fiber.
• In FISH, the marker is a DNA sequence that is
visualized by hybridization with a fluorescent
probe.

36. Fluorescent in situ hybridization (FISH)
Image
Source:
GENOMES 3

37. Sequence tagged sites (STS)
• STS mapping is the most powerful physical mapping
technique.
• Detailed Maps are generated by STS mapping.
• A sequence tagged site (STS) is a short DNA sequence,
generally between 100bp and 500bp in length.
• STS is easily recognizable and occurs once in the
chromosome or genome being studied.

38. Mapping of STS
• For mapping a set of STSs, a collection of overlapping
DNA fragments from a single chromosome or from
entire genome is needed.
• The data from which maps will be derived are obtained
by determining fragments which contain STSs.
• This can be done by hybridization technique and PCR.
• PCR is preferred because it is easier.

39. Mapping of STS
• If the two STSs are very
close then there is
always a chance that
they will be on the same
fragment.
• If they are farther apart
then they may
sometimes be on the
same fragment but
sometimes they will not.
• The data can therefore
be used for calculating
mapping distance
between two markers as
in linkage analysis.
Image Source:
GENOMES 3

40. Ways to obtain STSs
• STSs can be obtained in many ways but the
most common methods are:
– Expressed Sequence Tags (ESTs)
– SSLPs
– Random genomic sequences

41. Genetic Map VS Physical Map
• A genetic map is constructed using recombination
frequency calculated from the progenies whereas
physical mapping pertains to locating the position of
DNA sequences.
• A genetic map is an indirect method of locating the
positions of genes or DNA markers whereas physical
mapping is a direct method.
• The unit of measurement of map distance in genetic
map is cM whereas in physical map is the base pair.

42. Importance of Gene Mapping
• Gene map is the anatomy of human genome.
• It helps in analysis of the heterogeneity and
segregation of human genetic diseases.
• It helps to develop methods for gene therapy.
• It provides clinically useful information about
linkage

43. References
• Brown, T. A. (2007) GENOMES 3. 3rd Edition, Garland
Science Publishing
• Kant, Anil. (2008). Singh, R.K. Mishra G. P., Kant Anil,
Shashi Bala. Molecular markers in Plants. In: Molecular
Plant breeding; Principles and Applications. pp 79-96
Stadium press LLC, Texas, USA.. 77-79.
• Morgan, T. H. Random segregation versus coupling in
Mendelian inheritance. Science 34, 384 (1911)
• Zamir, D. & Tanksley, S.D. (1988) Mol Gen Genet 213:
254.

44. Thank You