GENE MAPPING & CLONING

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

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Contents
 Introduction
 Genome Mapping
 Genetic Mapping
 Physical Mapping
 Uses & Limitations of Gene Mapping
 Cloning
 Gene Cloning
 Reproductive Cloning
 Therapeutic Cloning
 References
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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.
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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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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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 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
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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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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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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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Physical Mapping
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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 11

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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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Genetic vs. Physical Distance
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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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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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 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.
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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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Types of Cloning
 Gene Cloning
 Reproductive Cloning
 Therapeutic Coning
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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.
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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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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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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

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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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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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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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 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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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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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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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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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.
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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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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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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.
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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.
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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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GENE MAPPING & CLONING

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