Somatic cell hybridization allows for gene mapping by fusing cells from different species. This results in heterokaryons containing a mixture of chromosomes. As the hybrid cells divide, they lose chromosomes at random. Observing which chromosomes are lost along with the corresponding phenotypes allows researchers to map genes to specific chromosomes. Key aspects include using cells deficient in different enzymes to select for viable hybrids and tracking chromosome and gene retention over multiple cell lines to assign locations. This technique is an important method for gene mapping.

1. Somatic Cell Hybridization

2. CELL to CELL hybridization
1. In vitro hybridization of somatic cells.
2. Method of mapping the location of a gene on a particular chromosome.
3. Heterotypic cell fusion
Homokaryon: when fusing cells are from the same species (intraspecies fusion)
Heterokaryon: when the fusing cells are from different species (interspecies fusion)
Binucleate Uninucleate

3. Cell Fusion
Spontaneous: natural fusion; but very rare
Induced: Polyethylene glycol (PEG), liposome, lysolethicin,
inactivated Sendai virus
Use:
1. Monoclonal antibody by hybridoma
2. Gene mapping

4. Cell fusion
Heterokaryon/hybrid
selection
Chromosome loss
Gene mapping by observing
the phenotype (eg. enzyme)

5. H R
H
R
H R
HR HR
Sendai virus mediated hybridization
Human (H) and Rodent cell
hybridize via a cytoplasmic
bridge induced by sendai
virus particle which gets
attached with plasma
membrane of one cell
Binucleate heterokaryon
During 1st mitosis after fusion all the
chromosomes from both parental
cells (H and R) are randomly attached
to mitotic spindle
Daughter cells contain single
nucleus with chromosomes
from each parent

6. Selection of hybrids
 Two important pathways for biosynthesis of nucleotides require for DNA synthesis i.e.
de novo pathway and salvage pathway.
 de novo pathway : major pathway by which nucleotides are synthesized from simple
sugars and amino acids or this pathway is blocked by antimetabolite aminopterin. If de
novo pathway is blocked, then second pathway called salvage pathway is turned on.
 HGPRT (Hypoxanthine-guanine phosphoribosyltransferase for synthesis of Guanindine)
and TK (Thymidine kinases for synthesis of deoxythymidine monophosphate) are 2
enzymes essential for Salvage pathway.
 Selection media is supplemented with Hypoxanthine, Aminopterin, and Thymdine (HAT
media).
 For fusion, parental human cell is deficient in HGPRT (HGPRT-) and rodent cell is
deficient in TK (TK-).
 Only hybrid cells with both functional genes will survive. Non-fused parental cells will
die

7. HGPRT-TK+
HGPRT-TK+ HGPRT- TK+
Dies
HGPRT+TK-
HGPRT+TK- HGPRT+ TK-
Dies
Selection on HAT media
HGPRT+TK-
HGPRT-TK+ HGPRT+ TK+
Survive

8. Chromosome loss in hybrids
Human + Mouse = loss of Human chromosome
Mouse + Rat = loss of Rat chromosome
Helps to assign the location of a gene on particular chromosome
Mapping of Gene A codes for protein/enzyme A (phenotype)
A
A
A
x
Gene A is located
on chromosome 4
1, 2, 3, 4

9. Hybrid
cell
line
Human chromosome present
(cytology)
Gene products
expressed
1 2 3 4 5 6 7 8 A B C D
21 p p p p X X X X - + - +
30 p p X X p p X X + - - +
41 p X p X p X p X + + - +
Gene A – chromosome 5
Gene B – chromosome 3
Gene C – chromosome 1-7
Gene D – chromosome 1

10. Molecular Markers

11. What is Marker?
Marker is a piece of DNA molecule that is
associated with a certain phenotype and
thus helps in identification of an
individual or a taxonomic group
AT
GC
AT
GC
AT
GC
CC
CC
Chr. 1
Chr. 2
Molecular Heterozygosity
Gene
Non coding

12. Molecular Markers
single nucleotide
polymorphisms
(SNP)
simple sequence
length polymorphisms
(SSLP)
DNA polymorphism (sequence and size variation)

13. difference in only 1 base [detectable]
1. 0.1% difference in genome between 2 individuals.
2. Human, 1 in every 300 to 1000 bases.
....AAGGCTCAT....
.... AACCGAGTA....
....AAAGCTCAT....
....TTTCGAGTA....
1. Silent SNP in gene - No effect on phenotype. Eg SNP in
recessive allele
2. SNPs in which one SNP allele causes a mutant phenotype
showing single-gene
inheritance.
3. Intergenic SNPs: non coding region
4. RFLP: SNPs located at a restriction enzyme's target site. In such
cases, there will be two RFLP "alleles”.
SNP

14. Restriction Fragment Length
Polymorphism (RFLP)
• Genomic DNA digested with Restriction
Enzymes
• DNA fragments separated via electrophoresis
and transfer to nylon membrane
• Membranes exposed to probes labelled with
P32 via southern hybridization
• Film exposed to X-Ray

15. 3 kb 2 kb
5 kb

16. Inheritance of RFLP markers
AA aa Aa
Useful in Paternity test, inheritance of disease
linked gene

17. Simple Sequence Length Polymorphisms
(SSLP)
• Different numbers of copies of adjacent multiple
repeats of short, simple DNA sequences (repetitive DNA
sequence)
• VNTR (Variable number tandem repeats)
• Application in Forensic science
1. Minisatellite
2. Microsatellite

18. variation in the number of tandem repeats of a repeating unit from 15 to 100
nucleotides long. In humans, the total length of the unit is from 1 to 5 kb.
Restriction cut on sites flanking the repeats followed by southern blot with probes
against the repeat.
Minisatellite or STR (short tandem repeat)

19. Microsatellite or SSR (simple sequence repeat)
variable numbers of tandem repeats of an even simpler sequence, generally a
dinucleotide.
5' C-A-C-A-C-A-C-A-C-A-C-A-C-A-C-A 3'
3' G-T-G-T-G-T-G-T- G-T-G-T-G-T-G-T 5'
M"is probably linked
in cis configuration
to the disease allele
P

20. Randomly Amplified Polymorphic DNA (RAPD)
• PCR based marker with 10-12 base pairs
• Random amplification of several fragments
• Random primers
• Amplified fragments run in agarose gel detected
by EtBr
• Unstable amplification leads to poor
repeatability

22. Amplified Fragment Length Polymorphism
(AFLP)
• Restriction endonuclease digestion of DNA
• Ligation of adaptors
• Amplification of ligated fragments
• Separation of the amplified fragments via
electrophoresis and visualization
• AFLPs have stable amplification and good
repeatability

23. Rare cutter: EcoRI
Frequent cutter: MseI
Adapter to seal the
sticky ends
Preselective: primer +
1 extra base
(stringent)
Selective: primer + 3
extra bases (even
more stringent)

24. Internal Simple Sequence Rpeat
(ISSR)
Region between two SSRs. ISSRs are amplified by PCR using microsatellite core sequences as
primers with a few selective nucleotides as anchors into the non-repeat adjacent regions
(16-18 bp).

25. Co-dominant markers Dominant markers
SSR
STR
RFLP
ISSR
RAPD
AFLP