Molecular markers are DNA sequences that can be used to identify differences between individuals. They are found at specific locations in the genome and can be used to track inheritance of traits. Common types include RFLPs, RAPDs, AFLPs, SSRs, and SNPs. RFLPs detect differences in fragment lengths after restriction enzyme digestion and probing. RAPDs use random PCR primers to amplify polymorphic loci. AFLPs combine restriction digestion and PCR to detect multiple loci. SSRs are co-dominant markers based on differences in repeated microsatellite sequences. Molecular markers are powerful tools for genetic mapping, diversity analysis, fingerprinting, and marker-assisted selection.
What is Genome,Genome mapping,types of Genome mapping,linkage or genetic mapping,Physical mapping,Somatic cell hybridization
Radiation hybridization ,Fish( =fluorescence in - situ hybridization),Types of probes for FISH,applications,Molecular markers,Rflp(= Restriction fragment length polymorphism),RFLPs may have the following Applications;Advantages of rflp,disAdvantages of rflp, Rapd(=Random amplification of polymorphic DNA),Process of rapd, Difference between rflp &rapd
RAPD markers are decamer DNA fragments.
RAPD is a type of PCR reaction.
as the name suggest it is a fast method when compared to the traditional PCR medthod.
Molecular Marker and It's ApplicationsSuresh Antre
Molecular (DNA) markers are segments of DNA that can be detected through specific laboratory techniques. With the advent of marker-assisted selection (MAS), a new breeding tool is now available to make more accurate and useful selections in breeding populations.
What is Genome,Genome mapping,types of Genome mapping,linkage or genetic mapping,Physical mapping,Somatic cell hybridization
Radiation hybridization ,Fish( =fluorescence in - situ hybridization),Types of probes for FISH,applications,Molecular markers,Rflp(= Restriction fragment length polymorphism),RFLPs may have the following Applications;Advantages of rflp,disAdvantages of rflp, Rapd(=Random amplification of polymorphic DNA),Process of rapd, Difference between rflp &rapd
RAPD markers are decamer DNA fragments.
RAPD is a type of PCR reaction.
as the name suggest it is a fast method when compared to the traditional PCR medthod.
Molecular Marker and It's ApplicationsSuresh Antre
Molecular (DNA) markers are segments of DNA that can be detected through specific laboratory techniques. With the advent of marker-assisted selection (MAS), a new breeding tool is now available to make more accurate and useful selections in breeding populations.
A physical map of a chromosome or a genome that shows the physical locations of genes and other DNA sequences of interest. Physical maps are used to help scientists identify and isolate genes by positional cloning.
According to the ICSM (Intergovernmental Committee on Surveying and Mapping), there are five different types of maps: General Reference, Topographical, Thematic, Navigation Charts and Cadastral Maps and Plans.
Techniques based on the principle of selectively amplifying a subset of restriction fragments from a complex mixture of DNA fragments obtained after digestion of genomic DNA with restriction endonucleases.
this presentation is about the molecular markers as we all know the molecular markers are the DNA sequences it can be easily detected and its inheritance is easily monitored.so the main basics of the molecular markers is the polymorphic nature so it can used as molecular markers.and this will gives you the idea about AFLP, RFLP, RAPD, SNPS,ETC.
A physical map of a chromosome or a genome that shows the physical locations of genes and other DNA sequences of interest. Physical maps are used to help scientists identify and isolate genes by positional cloning.
According to the ICSM (Intergovernmental Committee on Surveying and Mapping), there are five different types of maps: General Reference, Topographical, Thematic, Navigation Charts and Cadastral Maps and Plans.
Techniques based on the principle of selectively amplifying a subset of restriction fragments from a complex mixture of DNA fragments obtained after digestion of genomic DNA with restriction endonucleases.
this presentation is about the molecular markers as we all know the molecular markers are the DNA sequences it can be easily detected and its inheritance is easily monitored.so the main basics of the molecular markers is the polymorphic nature so it can used as molecular markers.and this will gives you the idea about AFLP, RFLP, RAPD, SNPS,ETC.
Comparative sequence studies of the repeat elements in diverse insect species can provide useful information on how to make use of them for developing abundant markers that can be used in those species;
$ At the moment, a total of 8 species are in genome assembly stages and another 35 are in progress for genome sequencing;
$ Different molecular marker systems in the field of entomology are expected to provide new directions to study insect genomes in an unprecedented way in the years to come
Molecular Breeding in Plants is an introduction to the fundamental techniques...UNIVERSITI MALAYSIA SABAH
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An honest effort to present molecular marker in easiest way both informative and conceptual. Hybridization based (non-PCR) and PCR based markers are discussed to the point with suitable diagram.
TYPES OF MOLECULAR MARKERS,ITS ADVANTAGES AND DISADVANTAGESANFAS KT
Types of molecular markers (genetics)
ITS ADVANTAGES AND DISADVANTAGES
What is a genetic marker?
RFLP: Restriction fragment length polymorphism
AFLP: Amplified fragment length polymorphism
RAPD: Random amplification of polymorphic DNA
ISSR: Inter simple sequence repeat
STR: Short tandem repeats
SCAR: Sequence characterized amplified region
SNP: Single nucleotide polymorphism
SSR: Simple sequence repeat
TYPES OF MOLECULAR MARKERS,ITS ADVANTAGES AND DISADVANTAGESANFAS KT
Types of molecular markers (genetics)
ITS ADVANTAGES AND DISADVANTAGES
What is a genetic marker?
RFLP: Restriction fragment length polymorphism
AFLP: Amplified fragment length polymorphism
RAPD: Random amplification of polymorphic DNA
ISSR: Inter simple sequence repeat
STR: Short tandem repeats
SCAR: Sequence characterized amplified region
SNP: Single nucleotide polymorphism
SSR: Simple sequence repeat
Molecular marker General introduction by K. K. SAHU Sir.KAUSHAL SAHU
Introduction
Molecular marker
Characterstics of molecular marker
Types of molecular marker
. Non PCR Based
. PCR Based
RFLP
RAPD
AFLP
SSR
SNP
Conclusion
References
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Molecular markers
1.
2. What are Molecular Markers?
Specific fragments of DNA that can be identified within the whole
genome.
Molecular markers are the general assays that allow detection of the
sequence differences between two or more individual.
Molecular markers are found at specific locations of the genome.
They are used to 'flag' the position of a particular gene or the
inheritance of a particular characteristic or desired characteristics
Definition
A molecular marker is a DNA sequence that is readily detected and
whose inheritance can easily be monitored .
3. Marker systems are tools which is used to mark a trait in living organism
MORPHOLOGICAL MARKER:
Classical markers
MOLECULAR MARKERS:
Variation in macro-molecules
BIOCHEMICAL MARKERS GENETIC MARKERS
ISOENZYME
PROTEIN
RFLP
AFLP
RAPD
They are protein produced by expression of gene
Depend upon sequence of DNA
•Low polymorphism
•Requires expression of trait / gene
•Dominance effect
•Expression sex limited
•Expressed late in life
4. DNA markers
Non-PCR Based,
RFLP- Restriction fragment length polymorphism.
PCR Based
RAPD- Random amplification of polymorphic DNA.
AFLP-Amplified fragment length polymorphism.
SCAR-Sequence characterize amplified region.
STS- Sequence tagged sites.
EST-Express sequence tags.
SNP-Single nucleotide polymorphism.
SSR-Simple sequence repeats
CAPS-Cleaved amplified polymorphic sequences.
5. it must be polymorphic.
Co-dominant inheritance.
A marker should be evenly and frequently
distributed throughout the genome.
It should be easy, fast and cheap to
detect.
It should be reproducible.
High exchange of data between
laboratories.
Properties of Ideal Genetic Marker
6. Feature RFLP RAPD AFLP SSR or
Microsatellite
DNA required (µg) 10 0.02 0.5-1.0 0.05
DNA quality High High Moderate Moderate
PCR-based No Yes Yes Yes
No. of polymorphic loci
analysed
1.0-3.0 1.5-50 20-100 1.0-3.0
Ease of use Not easy Easy Easy Easy
Reproductibily High Unreliable High High
Development cost Low Low Moderate High
Cost per analysis High Low Moderate Low
Table 1 : Comparision of the most broadly used techniques
of molecular markers
Cont…..
7. Definition
The variation(s) in the length of DNA fragments produced by a specific
restriction endonuclease from genomic DNA s of two or more
individuals of a species
RFLP
8. Principle
Restriction fragment length polymorphism (RFLP) technology was first developed in
the 1980s for use in human genetic applications and was later applied to plants.
By digesting total DNA with specific restriction enzymes, an unlimited number of
RFLPs can be generated.
RFLPs are relatively small in size and are co-dominant in nature.
If two individuals differ by as little as a single nucleotide in the restriction site, the
restriction enzyme will cut the DNA of one but not the other. Restriction fragments of
different lengths are thus generated.
All RFLP markers are analyzed using a common technique. However, the analysis
requires a relatively complex technique that is time consuming and expensive.
9. The hybridization results can be visualized by
1. Autoradiography (if the probes are radioactively labeled), or
2. Chemiluminesence (if non-radioactive, enzyme-link methods are used for
probe labeling and detection).
Any of the visualization techniques will give the same results. The visualization
techniques used will depend on the laboratory condition
Principle
11. -simple method as no sequence specific information is required
-codomonant markers
-it is not depend on PCR
-it required large amount of highly pure DNA
-it require constant supply of probes
-it is laborious to identify suitable markers
-it is time consuming
-it requires expertise in autoradiography
Advantages
Disadvantages
12. Applications of molecular markers
►MEASURE OF GENETIC DIVERSITY
►FINGER PRINTING
►GENOTYPIC SELECTION
►GENOTYPIC PYRAMIDYING AND
INTROGRESSION
►INDIRECT SELECTION USING QUANTITATIVE
TRAITS LOCI (QTLS)
►MARKER-ASSISTED SELECTION
►IDENTIFICATION OF GENOTYPE
13. Any DNA segment that is amplified using short oligodeoxynucleotide primer
of arbitrary nucleotide sequence (amplifier) and polymerase chain reaction
procedure (Khal, 2001)
RAPD also known as,
AP-PCR(Arbitrarily primed PCR),
DAF (DNA amplification fingerprinting)&
MAAP(Multiple arbitrary amplicon profiling)
RAPD are a dominant marker system
Definition
RAPD
14. 1. RAPDs are produce by PCR using genomic DNA and arbitrary primers
2. Taq polymerase is used to amplify DNA segment between closely spaced
sequence (< 2kb) and complementary to the short random oligomers (typically
10-mers)
3. RAPD polymorphism result from change in the primer-binding site in the DNA
sequence
Principle
15. In variety A there are 4 primer binding sites resulting in two RAPD products, variety
B lacks one of the binding sites resulting in only one RAPD marker being produced
19. Protocol
1) Master Stock Mixture
2) Add 25µl of master mix to 5µl of your DNA in a sterile tube
Note: In each PCR run you conduct, include 2 sample, one of control DNA
without primer (3µl DNA), and one sample without DNA (5µl ddH2O)
20. -need small amount of DNA
-it involves non-radioactive assay
-it does not required specific probe libraries
-it provide quick and efficient screening for DNA
sequence based on polymorphism at many loci
-it is inherited as dominant traits
-there is a bands due to relatively short primer
-the production of non-parental bands in the offspring of
known pedigree warrants its use with extreme care
-it is sensitive to change in PCR conditions
Advantages
Disadvantages
22. AFLP
Definition
Any difference between corresponding DNA fragment from two
organisms A & B that is detected by amplified restriction length
polymorphism technique
23. Principle
1. The amplified fragment length polymorphism technique combines components
of RFLP analysis with PCR technology.
2. Total genomic DNA is digested with a pair of restriction enzymes normally a
frequent and rare cutter.
3. Adaptors of known sequence are then ligation to the DNA fragments.
4. Primer complementary to the adaptors are used to amplify the restriction
fragments.
5. The PCR amplified fragments can then separated by gel electrophoresis and
banding patterns visualized.
6. A range of enzymes and primer are available to manipulate the complexity of
AFLP fingerprint to suit application
24. Principle
Genomic DNA
For restriction digestion we use two type of cutter i.e
•Rare cutter (6bp) EcoRI
•Frequent cutter (4bp) MseI
Interstitial cohesive ends
Adaptor ligation
PCR amplification using EcoRI/MseI
25. -extremely sensitive
-it has a wide scale applicability
-it discriminates heterozygotes from
homozygotes when a gel scanner is used
-used for mapping
-it is highly expensive
-it required more DNA than RAPD
-it required experience of sequencing gels
Advantages
Disadvantages
28. Microsatellites can be amplified for identification by the polymerase
chain reaction (PCR) process, using the unique sequences of flanking
regions as primers
DNA is repeatedly denatured at a high temperature to separate the
double strand, then cooled to allow annealing of primers and the
extension of nucleotide sequences through the microsatellite.
This process results in production of enough DNA to be visible
on agarose or polyacrylamide gels.
With the abundance of PCR technology, primers that flank
microsatellite loci are simple and quick to use, but the development of
correctly functioning primers is often a tedious and costly process.
Principle
29.
30. -simple and easy to use
-easy to detect via PCR
-co-dominant marker
-perfectly suited for used in map-based cloning
-cost is higher for establishing polymorphic primer sites
and investment in the synthesizing the oligonucleotides
-initial identification, DNA sequence information
necessary
Advantages
Disadvantages
31. Application of SSR
Assessment of genetic variability and characterization of germplasm.
Identification and fingerprinting of genotypes.
Estimation of genetic distances between population, inbreeds and
breeding material.
Marker assisted selection.
Identification of sequence of useful candidate genes
34. M R H H S R H H S R H H S H R R H R H
Fig: Identification of RAPD marker link to brown plant hoper resistance
gene in rice
June et al.,2003
3
35. RAPD-ANALYSIS OF GENETIC VARIATION OF FOUR IMPORTANT RICE
VARIETIES USING RAPD PRIMERS
Amplified RAPD patterns of OPR1
M - 1 Kb DNA Ladder
1 - ADT38
2 - ASD16
3 - IR20
4 - PONNI
Amplified RAPD patterns of OPR2.
Mani et al. (2010)Tamil Nadu
Con..
2
36. UPGMA dandogram based on Nei’s
(1978) original measure of genetic
distance, summarizing the
data on differentiation between four
samples of O. sativa genotypes according
to RAPD analysis.
Genetic distance between O. sativa populations of four different
rice varieties based on Nei’s 1978 measures of genetic distance.
PONNI IR-20 ADT38 ASD16
PONNI 0.3913 1.7776 1.02564
IR-20 0.3913 1.60944 1.95601
ADT38 1.77767 1.60944 0.8574
ASD16 1.02564 1.95601 0.8574
37. 1 2 3 4 5
Fig: Molecular mapping of fertility restorer gene in basmati rice
using micro satellite marker.
Delhi Mishra et al.2001
A Rice microsatellite marker RM 258 identified to be linked with fertility
restorer gene in PRR- 78 using bulk segregant analysis.
DNA marker (lane 1). Restorer line PRR 78 (lane 2).
CMS line IR 58025 (lane 3).
Fertile bulk showing heterozygous pattern (lane 4).
Sterile bulk showing homozygous pattern (lane 5)
38.
39.
40. Fig. 7 : Hybrids identification by using seed protein markers
Bhubaneshwar Panigrahi et al., 2001
45. Advantages:
Highly variable
Easy to detect via PCR
Fast evolving
Co dominant
Disadvantage:
Relatively expensive and time consuming to develop
Initial identification, DNA sequence information necessary
46. 46
PCR: Polymerase Chain Reaction
“Amplify” DNA by in-vitro (in plastico) synthesis
Key requirements:
enzyme: Taq DNA polymerase, not denatured at high
temps used to denature DNA
primers: short (~ 20 b) oligonucleotides bind to
denatured DNA, required to start DNA synthesis