This document discusses gene mapping and gene cloning. It defines gene mapping as identifying the locus and distance between genes using genetic or physical mapping techniques. Gene cloning involves inserting a fragment of DNA containing a gene into a cloning vector, which is then propagated in bacteria to make multiple copies. The document provides detailed descriptions of genetic mapping, physical mapping, gene cloning techniques like transformation, PCR cloning, and their applications and limitations.

1. Name: C.Bindu Lasya
Gene Mapping and Gene cloning
M.Pharmacy
Department of Pharmacology
ANURAG UNIVERSITY

2.  Gene Mapping describes the methods used to
identify the locus of the gene and the distance
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 genome maps, and mapped the same way
as any other markers in the construction of genome
maps, and mapped the same way as any other
markers.

3.  Genetic mapping is based on the 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
(pedigree).
 Physical mapping uses molecular biology
techniques to examine DNA molecules directly in
order to construct maps showing the positions of
sequence features, including genes.

4.  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 qualify 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.

5.  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 neighboring markers.
 The entire process is then repeated by looking at more
markers which target that region to map the gene
neighborhood 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.

7.  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 chromosome.
 Sequence tagged site (STS) mapping, in which
the positions of short sequences are mapped by PCR
and/or hybridization analysis of genome fragments.

8.  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.

10.  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.

12.  Identify genes responsible for diseases.
 Heritable diseases
 Cancer
 Identify genes responsible for traits.
 Plants or Animals
 Disease resistance
 Meat or Milk Production

13.  The Human Genome Project (HGP) is an
international scientific research project with the goal
of determining the sequence of chemical base pairs
which make up human DNA, and of identifying and
mapping all of the genes of the human genome from
both a physical and a functional standpoint.
 The Human Genome Project originally aimed to map
the nucleotides contained in a human haploid
reference genome (more than three billion). The
"genome" of any given individual is unique; mapping
the "human genome" involves sequencing multiple
variations of each gene. In May 2016, scientists
considered extending the HGP to include creating a
synthetic human genome.

14.  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.
 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.

15.  Gene Cloning:- The insertion of a fragment of
DNA carrying a gene into a cloning vector and
subsequent propagation of recombinant DNA
molecules into many copies is known as gene
cloning.
 It involves using bacteria to make multiple copies of
a gene.
 Foreign DNA is inserted into a plasmid, and the
recombinant plasmid is inserted into a bacterial cell.
 Reproduction in the bacterial cell results in cloning of
the plasmid including the foreign DNA.
 This results in the production of multiple copies of a
single gene.

16.  Gene manipulation is defined as the formation of a
new combination of a heritable material by the
insertion of nucleic acid molecules ( DNA molecules)
into bacterial plasmid or any other vector so as to
allow their incorporation in to the host organism, in
which they are capable of containing propagation.

19. Transformation:
 A bacterium takes up DNA from the medium.
 Recombination takes place between introduced
genes and the bacterial chromosome.

20.  Methods:
1) Electroporation
2) Freeze Thaw Method
3) Calcium Chloride Mediated Transformation
4) Calcium Phosphate Mediated Transformation

21.  Treatment with calcium chloride in the early log
phase of growth for Competence
 Bacterial cell membrane is permeable to chloride
ions,
but is non-permeable to calcium ions.

22. As the chloride ions enter the
cell, water molecules accompany
the charged particle.
Influx of water causes the cells
to swell and is necessary for the
uptake of DNA.
The exact mechanism of this
uptake is unknown.
DNA of interest is then added
to the cells.

23. Calcium chloride treatment be
followed by addition of DNA of
interest then by heat.
The heat shock step is necessary
for the uptake of DNA.
Temperatures > 42degC Bacteria’s
ability to uptake DNA reduces .
Extreme temperatures: Bacteria
dies.
After the heat shock step intact
plasmid DNA molecules replicate in
bacterial host cells

24.  To help the bacterial cells recover from the heat shock cells
are briefly incubated with nonselective growth media.

25. As the cells recover, plasmid
genes are expressed
• Bacterial colonies selected
using antibiotic selection
techniques

27.  Lose of antibiotic resistance.
 Colony hybridization.
 Immuno chemical method.

28.  PCR cloning differs from traditional cloning in that
the DNA fragment of interest, and even the vector,
can be amplified by the Polymerase Chain Reaction
(PCR) and ligated together, without the use of
restriction enzymes.
 PCR cloning is a rapid method for cloning genes, and
is often used for projects that require higher
throughput than traditional cloning methods can
accommodate.
 It allows for the cloning of DNA fragments that are
not available in large amounts.
 Typically, a PCR reaction is performed to amplify the
sequence of interest, and then it is joined to the
vector via a blunt or single-base overhang ligation
prior to transformation.

29.  Early PCR cloning often used Taq DNA Polymerase to
amplify the gene. This results in a PCR product with a
single template-independent base addition of an adenine
(A) residue to the 3' end of the PCR product through the
normal action of the polymerase.
 These "A-tailed" products are then ligated to a
complementary T-tailed vector using T4 DNA ligase,
followed by transformation.

31.  High-fidelity DNA polymerases are also now routinely used
to amplify sequences with the PCR product containing no 3'
extensions.
 The blunt-end fragments are joined to a plasmid vector
through a typical ligation reaction or by the action of an
"activated" vector that contains a covalently attached
enzyme, typically Topoisomerse I, which facilitates the
vector:insert joining.
 Some PCR cloning systems contain engineered "suicide"
vectors that include a toxic gene into which the PCR
product must be successfully ligated to allow
propagation of the strain that takes up the recombinant
molecule during transformation.
 A typical drawback common to many PCR cloning
methods is a dedicated vector that must be used.

32.  These vectors are typically sold by suppliers, like NEB, in a
ready-to-use linearized format and can add significant
expense to the total cost of cloning. Also, the use of
specific vectors restricts the researcher's choice of
antibiotic resistance, promoter identity, fusion partners,
and other regulatory elements.

33.  Advantages:
 High efficiency, with dedicated vectors.
 Amenable to high throughput.
 Disadvantages:
 Limited vector choices.
 Higher cost.
 Lack of sequence control at junction.
 Multi-fragment cloning is not straight forward.
 Directional cloning is difficult.