Microteaching on terms used in filtration .Pharmaceutical Engineering
GENE MAPPING & CLONING
1. Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Gene Mapping & Cloning
A Seminar as a part of curricular requirement for
Master of Pharmacy,
I Year - I semester
Presented by
Mary Vishali
(20L81S0104)
Dept of. Pharmacology
Under the guidance of
Dr. K. Soma Sekhar Reddy M.Pharm, Ph.D.
Associate Professor & HOD Pharamacology
2. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Contents
Introduction
Genome Mapping
Genetic Mapping
Physical Mapping
Uses & Limitations of Gene Mapping
Cloning
Gene Cloning
Reproductive Cloning
Therapeutic Cloning
References
2
3. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Introduction
Gene mapping describes the methods used to identify the locus of a
gene and the distances between genes.
The essence of all genome mapping is to place a collection of
molecular markers onto their respective positions on the genome.
Molecular markers come in all forms. Genes can be viewed as one
special type of genetic markers in the construction of genome maps,
and mapped the same way as any other markers.
3
4. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Genome Mapping
Genetic mapping is based on the use of genetic techniques to
construct maps showing the positions of genes and other sequence
features on a genome.
Genetic techniques include cross-breeding experiments or,
Case of humans, the examination of family histories
(pedigrees).
Physical mapping uses molecular biology techniques to examine
DNA molecules directly in order to construct maps showing the
positions of sequence features, including genes.
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5. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Genetic Mapping
The first steps of building a genetic map are the development of genetic
markers and a mapping population. Since the closer the two markers are
on the chromosome, the more likely they are to be passed on to the next
generation together, therefore the "co-segregation" patterns of all
markers can be used to reconstruct their order. The genotypes of each
genetic marker are recorded for both parents, and in each individual in
the following generations. The quality of the genetic maps is largely
dependent upon these two factors: the number of genetic markers on the
map and the size of the mapping population.
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6. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
In gene mapping, any sequence feature that can be
faithfully distinguished from the two parents can be used as a
genetic marker. Genes are represented by "traits" that can be
distinguished between two parents.
Their linkage with other genetic markers are calculated same way
as if they are common markers and the actual gene loci are then
bracketed in a region between the two nearest neighbouring
markers.
The entire process is then repeated by looking at more markers
which target that region to map the gene neighbourhood
6
7. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
to a higher resolution until a specific causative locus can be identified.
This process is often referred to as "positional cloning", and it is used
extensively in the study of plant species.
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8. RIPER
AUTONOMOUS
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Physical Mapping
Restriction mapping, which locates the relative positions on a DNA
molecule of the recognition sequences for restriction endonucleases;
Fluorescent in situ hybridization (FISH), in which marker locations
are mapped by hybridizing a probe containing the marker to intact
chromosomes;
Sequence tagged site (STS) mapping, in which the positions of short
sequences are mapped by PCR and/or hybridization analysis of
genome fragments.
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9. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Physical maps
Physical maps can be generated by aligning the restriction maps of
specific pieces of cloned genomic DNA (for instance, in YAC or
BAC vectors) along the chromosomes.
These maps are extremely useful for the purpose of map-based gene
cloning.
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10. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Physical Mapping
10
11. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 11
12. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Genetic vs. Physical Distance
Map distances based on recombination frequencies are not a direct
measurement of physical distance along a chromosome.
Recombination “hot spots” overestimate physical length.
Low rates in heterochromatin and centromeres underestimate actual
physical length.
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13. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Genetic vs. Physical Distance
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14. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Uses of Gene Mapping
Identify genes responsible for diseases.
Heritable diseases
Cancer
Identify genes responsible for traits.
Plants or Animals
Disease resistance
Meat or Milk Production
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15. RIPER
AUTONOMOUS
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Limitations
A map generated by genetic techniques is rarely 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 .
Genes that are several tens of kb apart may appear at the same
position on the genetic map.
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16. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Genetic maps have limited accuracy .
Presence of recombination hotspots means that crossovers are
more likely to occur at some points rather than at others.
physical mapping techniques has been developed to address this
problem.
16
17. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
What is Cloning
A clone is a genetically identical copy of an organism, and it may be
naturally occurring or created in the lab. Through the process of
asexual reproduction, organisms such as bacteria (and some plants)
create offspring that are genetically identical to the parent. Modern
genetic technology can also be used to create clones.
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18. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Types of Cloning
Gene Cloning
Reproductive Cloning
Therapeutic Coning
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19. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Gene Cloning
Making multiple copies of a single gene.
The insertion of fragment of DNA carrying a gene into a cloning vector
and subsequent propagation of recombinant DNA molecule into many
copies is known as gene cloning.
19
20. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 20
21. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Basic Steps of Gene Cloning
Construction of recombinant DNA molecule.
Transport of the recombinant DNA to the host cell.
Multiplication of recombinant DNA molecule.
Division of the host cell.
Numerous cell division resulting in the clone.
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22. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Gene cloning requires specialized tool and
techniques
Vehicles: the central compensation of a gene cloning experiments is the
vehicle which transport the gene into the host cell and is responsible for
its replication. To act as a cloning vehicle a DNA molecule must be
capable of entering a host cell and once inside replicating to produce
multiple copies of itself.
Vector: A DNA molecule capable of replication in a host organism into
which gene is inserted to construct a recombinant DNA molecule.
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23. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Characteristic of a Vector
It must be able to replicate.
There must be some way to introduce vector DNA into cell.
There must be some means of detecting its presence, preferably
planting test in petri dishes.
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Cloning vector components
Ori
ampR gene
lacZ gene
Restriction gene
24. RIPER
AUTONOMOUS
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Plasmid
Characteristics
They are small and contains 3 – 5 kb of DNA.
They are contain a suitable markers (antibiotic resistant).
They contains suitable restrictions sites that can be used for insertion
DNA fragments for cloning.
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25. RIPER
AUTONOMOUS
NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Cloning Vector
A plasmid into which the gene of interest is introduced.
Contains a number of specific gene useful in selection.
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26. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Cloning Vector Components
Ori
amp R gene
lacZ gene
Restriction gene
Replication origin (ori):
Allow plasmid to replicate in the host cell
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27. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Antibiotic resistance (ampR) gene: allow cells to be resistance to
ampicillin (an antibiotic).
Selection for host cells that have resistance.
This, selection for transformation
β – galactosidase (lacZ) gene: enzyme produced will change a clear
substrate called X-gal into a blue product.
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28. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 28
29. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Step – 1 Forming Recombinant DNA
Where would you insert the DNA of interest so that you can “see” it
in the bacterial cell (Assume cells are grown in X-gal)
Ligate the gene of interest into the vector such that it interrupts the
lacZ gene.
Thus β- Galactosidase is not made.
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30. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Step – 2 Transformation
Transformation recombinant DNA into bacterial cell.
As bacterial cell multiply, the gene of interest will be replicated
with each other.
Bacterial grown in flakes of liquid medium.
Incubate at optimal growing temperature.
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31. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Step – 3 Selection
Selection: identify colonies of bacteria containing the recombination
DNA with gene of interest.
Possible bacterial clone products :
A. Bacterial without vector
B. Bacteria with vector without gene
C. Bacterial with vector with the gene of interest
Planting taking a sample of the bacteria and growing them on plates.
Plates have a medium containing: antibiotics
X-gal
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32. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Selection Mechanism: Antibiotic Resistance
Selection for bacterial clones that contains a vector (select for
proper transformation).
Bacteria are grown on Petri plate containing a specific antibiotics.
Vector confers antibiotics resistance to bacteria because the vector
contains an ampR gene.
Only bacterial cells that properly transformed the vector will live
and grow on the plate.
32
33. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Cloning Application: Bt Plants
Bacillus Thuringiensis a bacterium used a biological pesticide.
Bt gene is cloned into plants so that they will be resistant to pests.
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34. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Reproductive Cloning
Reproductive cloning involves crating and animal that is genetically
identical to a donor animal through somatic cell nuclear transfer.
In reproductive cloning, the newly created embryo is placed back
into the uterine environment where it can implant and develop.
Ex. The sheep Dolly
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35. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Therapeutic Cloning
In therapeutic cloning an embryo is created in a similar way to that
of the reproductive cloning but the resulting cloned cells remain in a
dish in the lab, they are not implanted into a female uterus.
35
36. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Cloning Application: Flavr savr Tomatoes
First genetically modified procedure.
Genetically modified tomatoes that suppressed a gene responsible
for fruit ripening.
Process required cloning the gene and transformation a reserve
orientation copy which would have inhibitory effects.
36
37. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
References
T.A Brown, Gene Cloning and DNA Analysis an Introduction, Sixth
Edition.
Anil G. Menon, Charles A. Klanke, and Yan Ru Su, Identification of
Disease Genes By Positional Cloning.
Rita M. Cantor, Analysis of Genetic Linkage.
Huntington’s Disease Genetics Department of neurology, Boston
University School of Medicine, Boston, Massachusetts.
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38. Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721