genetic engineering of plants

1. GENE MAPPING
Afsath.B
M Pharm 1st year
APPLIED PHARMACOGNOSY

2.  GENE-a unit of heredity which is transferred from a parent to offspring and is held to
determine some characteristic of the offspring.
 GENOME-the complete set of genes or genetic material present in a cell or organism.
 GENE MAPPING -Is the methods used to identify the locus of a gene and the distances
between genes.
Can offer firm evidence that a disease transmitted from parent to child is linked to one
or more genes. Mapping also provides clues about which chromosome contains the gene and
precisely where the gene lies on that chromosome.
Genetic maps have been used successfully to find the gene responsible for relatively
rare, single-gene inherited disorders such as cystic fibrosis and muscular dystrophy. Genetic
maps are also useful in guiding scientists to the many genes that are believed to play a role in
the development of more common disorders such as asthma, heart disease, diabetes, cancer, and
psychiatric conditions.
 Goal of gene mapping is to study the regulation and expression of genes.
 GENE EXPRESSION- is the process by which information from a gene is used in the
synthesis of a functional gene product
 Genes are expressed in 3 ways:-
 Visible
 Biochemical

3. TYPES OF GENOME MAPPING
LINKAGE MAPPING PHYSICAL MAPPING
• Arrangement of gene & DNA markers on a
chromosome
• Relative distance between position on a genetic map
are calculated using recombinant frequencies
• provide physical distance between landmarks on the
chromosome, ideally measured in nucleotide bases.
• Map is created by fragmenting the DNA molecule
using restriction enzymes and then looking for
overlaps
• TYPES-chromosomal or cytogenetic maps, radiation
hybrid (RH) maps, and sequence maps
• Done by means of a marker
• Eg. DNA finger printing

4. MARKERS
A genetic marker is a gene or DNA sequence with a known location on a chromosome that can be used to identify
individuals or species. Also used to find out relationship between genes.
a) Genes
b) DNA markers
1. RFLPs (Restriction Fragment Length Polymorphism)
2. SNPs (Single Nucleotide Polymorphism)
3. AFLP (Amplified Fragment Length Polymorphism)
4. RAPD (Random Amplification of Polymorphic DNA)
5. SSLPs (Single/ Simple Length Polymorphism)
6. Microsatellite/STRs (Simple Tandem Repeats)/Simple Sequence Repeats (SSR)
7. Mini-satellites/VNTRs (Variable Number of Tandem Repeats)
RFLPs (Restriction Fragment Length Polymorphism)
■ is a molecular marker
■ Most RFLP markers are co-dominant (both alleles in heterozygous sample will be detected) and highly locus-
specific.
■ Sensitive
■ difference in homologous DNA sequences that can be detected by the presence of fragments of different lengths
after digestion of the DNA samples by means of Restriction enzyme

5. STEPS IN RFLP ANALYSIS……..
1. Isolate DNA.
2. Perform PCR.
3. Perform Restriction Digestion.
4. Prepare Sample for Analysis.
5. Perform Capillary Electrophoresis.
6. Analyse Data.
LIMITATION
• Require high quality & quantity DNA
• Low polymorphism
• Require specific probe library dvpmt
• Require radioactive labelled probe
• Laborious and time consuming

6. ADVANTAGES
• High reproducibility
• Show codominant allele
• Detect coupling phase of DNA
• Reliable marker in linkage and breeding
analysis
• Easily determine a linked trait present in both
homozygous and heterozygous

7. RAPD (Random Amplified Polymorphic DNA)
o PCR based technology
o Procedure detect nucleotide sequence polymorphism in DNA
o Which randomly amplify anonymous segment of nuclear DNA with an identical pair of 8-10 bp
in length
o Because primers are short and relatively low annealing temperature (often 36 -40˚C) are used
o Need only one primer instead of set of primers for amplification
o Does not require any prior information about DNA sequence of desired organism
o The identical decamer (10-mer) will or will not amplify a segment of DNA, depending on
position that are complimentary to primer sequence.
o Amplified product are separated on agarose gel in presence of ETBR and view under UV
ADVANTAGE
• Quick & easy to assay
• Low quantities of template DNA required
• Dominant marker
• In expensive

8. DISDVANTAGES
• Low reproducibility
• Highly sensitive & complicated
procedure
• PCR cycling greatly influence the
out come
• Mismatch between primer &
template may result in total absence
of PCR product
APPLICATION
• Gene mapping
• DNA amplification finger printing
• Study of closely related species

9. AFLP (Amplified Fragment Length Polymorphism)
• Also known as Selective Restriction Fragment Amplification (SRFA)
• is a PCR-based tool used in genetics research, DNA fingerprinting, and in the practice of genetic
engineering
• This technique used to detect polymorphisms in DNA when no information about the genome is
known.
• AFLP-PCR is a highly sensitive method for detecting polymorphisms in DNA
• A DNA polymorphism is any difference in the nucleotide sequence between individuals( due to
mutation or rearrangement either at or in between the primer binding site). These differences can
be single base pair changes, deletions, insertions, or even changes in the number of copies of a
given DNA sequence
• uses restriction enzymes to digest genomic DNA, followed by ligation of adaptors to the sticky
ends of the restriction fragments.
• AFLP is more efficient than RFLP & RAPD in detection of polymorphism and has high
reproducing ability.
• well known as dominant markers.
• involves both the previous mapping basics such as restriction digestion of RFLP and
PCR amplification of RAPD.

10. WHY PCR USED IN MAPPING???????
■ Gene as a marker has limitation
■ Gene occupy small portion or space of genome
■ Every gene cannot be distinguished easily
■ So map based on gene is not detailed
■ There comes need of other marker called as DNA marker

11. STEPS IN AFLPANALYSIS……..
1. Digestion of total cellular DNA with one or more restriction enzymes and ligation of restriction
half-site specific adaptors to all restriction fragments.
2. Pre-selective amplification
3. Selective amplification of some of these fragments with two PCR primers that have
corresponding adaptor and restriction site specific sequences.
4. Electrophoretic separation of amplicons on a gel matrix, followed by visualisation of the band
pattern.

12. ADVANTAGES &APPLICATION
• Reproducibility
• Resolution
• Sensitivity
• High genomic abundance
• Has the capability to amplify between 50 and 100 fragments at one time.
• No prior sequence information is needed for amplification
• Beneficial in the study of taxa including bacteria, fungi, and plants
• Detect various polymorphisms in different genomic regions simultaneously
• Widely used for the identification of genetic variation in strains or closely related species of
plants, fungi, animals, and bacteria
DISADVANTAGES
• Need purified, high molecular weight DNA
• These are dominant markers
• Abundance of data

13. SNPs (Single Nucleotide Polymorphism)
• SNPs is a variation in a single nucleotide that occurs at a specific position in the genome
• Polymorphisms resulting from point mutations are the most abundant polymorphism in organisms
• SNPs marker development can be automated, and have the power to reveal hidden polymorphism not
detected with other markers and methods.
• SNPs do not necessary cause disease, but they can help to determine the likelihood that someone will
develop a particular illness
• Sometime: wide range of human diseases result from SNPs are; e.g. sickle-cell anaemia, β-thalassemia and
cystic fibrosis
APPLICATION
• Determine whether a genetic variant is associated with a disease or trait (disease diagnosis)
• Finding predisposition to disease4
• In discovery & development
• In drug responses

14. SSLP (Simple Sequence Length Polymorphism)
• SSLP is a type of polymorphism: a difference in DNA sequence amongst individuals
• SSLPs are repeated sequences over varying base lengths in intergenic regions of deoxyribonucleic acid
(DNA)
• Variance in the length of SSLPs can be used to understand genetic variation between two individuals in a
certain species
MINI-SATELLITE/VNTRs
• Is a tract of repetitive DNA in which certain DNA (ranging in length from 10-100 base pairs)

15. Microsatellite/ STRs
■ Is a tract of repetitive DNA in which certain DNA motifs (ranging in length from 2–5 base pairs) are
repeated, typically 5–50 times.
■ Microsatellites are often referred to as short tandem repeats (STRs) by forensic geneticists, or as simple
sequence repeats (SSRs) by plant geneticists
■ Microsatellites are located in regulatory flanking or attacking or intronic regions of genes, or directly in
codons of genes
■ STRs vary from person to person & Microsatellite mutations in such cases can lead to phenotypic changes
and diseases

16. APPLICATION OF MARKERS
1. Agricultural Applications
Knowledge of the genetic maps of plants and animals leads to the development of agricultural crops
and animal breeds that are more nutritious, productive and can better resist diseases, insects and drought.
Researchers can breed special plants that help clean up wastes that are difficult to break down.
2. Energy and the Environment
Genetic maps of microbes enable researchers to harness the power of bacteria for producing energy
from bio-fuels, reducing toxic waste, and developing environment-friendly products and industrial processes.
3. Forensics
You are already familiar with the use of genetic mapping in crime investigations, paternity tests, and
identification. The technique can also be used in organ transplants to achieve better matches between
recipients and donors, thus minimizing the risks of complications and maximizing the use of donated healthy
organs, a scarce resource. For more delectable applications, genetic mapping can authenticate the origins of
consumer goods like caviar, fruits, and wine or the pedigree of livestock and animal breeds.