1. Applied Molecular Genetics Molecular Marker and Technique
Dr. Manish Dev Sharma

2. Genetic Markers
Genetic variation, in the form of multiple alleles of many genes, occurs between organisms of a population and also within and between natural populations
Such genetic differences are due to DNA markers or (DNA polymorphisms; poly = many, morph = form) that are present in the genomes of organisms
Genetic (or DNA) markers are detected by direct analysis of the DNA
often, this requires genomic DNA that is fragmented into smaller pieces that are easier to handle and manipulate to reveal genetic differences, i.e. DNA sequence variation
Genetic markers can be detected at three levels,
phenotype (morphological, e.g. phenotype such as dwarfism, albinism, etc)
differences in proteins (biochemical, e.g. isozymes – different forms of the same protein)
differences in the nucleotide sequence of DNA (molecular)
Dr. Manish Dev Sharma, Assistant professor, Department of Life Sciences, SGRRITS, Dehradun

3. Properties of a good genetic marker
Polymorphic
to allow the differentiation of chromosomes carrying the mutant gene from those carrying the normal gene
polymorphisms are also used to measure genetic diversity
Reproducible – exchange of data between laboratories
Codominant inheritance – to allow the discrimination of homozygotes from heterozygotes
Evenly and frequently distributed throughout the entire genome
Discriminating – to allow the detection of genetic differences between closely related individuals
Not subjected to environmental influences/developmental stages
Evolutionary neutral – not under selection pressure
Inexpensive – easy, fast and cheap application
Dr. Manish Dev Sharma, Assistant professor, Department of Life Sciences, SGRRITS, Dehradun

4. Types of Genetic Markers
Single Nucleotide Polymorphism (SNP)
Restriction Fragment Length Polymorphism (RFLP)
Random Amplified Polymorphic DNA (RAPD)
Amplified Fragment Length Polymorphism (AFLP)
Simple Sequence Repeats (SSR)
Variable Number Tandem Repeats (VNTR)
Cleaved Amplified Polymorphic Sequence (CAPS)
Sequence Characterized Amplified Region (SCAR)
Expressed Sequence Tag (EST)
Tandem Number Repeats
Dr. Manish Dev Sharma, Assistant professor, Department of Life Sciences, SGRRITS, Dehradun

5. Single Nucleotide Polymorphism (SNP)
SNP (pronounced “snip”) - polymorphisms caused by point mutations that give rise to different alleles containing alternative bases at a given nucleotide position within a locus
They are the most abundant polymorphism in any organism. For example,
mouse – 1 SNP per 104 bp
humans – 1 SNP per 1250 bp
SNPs are usually restricted to one of two alleles and, thus, are referred to as biallelic
5’-AGTCTAGCTCATGGCTA-3’
3’-TCAGATCGAGTACCGAT-5’
5’-AGTCTAGCTCATGACTA-3’
3’-TCAGATCGAGTACTGAT-5’
Gene sequence level
5’-AGTCTAGCTCATGACTA-3’
5’-AGTCTAGCTCATGGCTA-3’
Gene 1
5’-AGTCTAGCTCATGGCTA-3’
5’-AGTCCAGCTCATGGCTA-3’
Gene 2
Dr. Manish Dev Sharma, Assistant professor, Department of Life Sciences, SGRRITS, Dehradun

6. SNPs display codominant inheritance
900 bp
350 bp
550 bp 5’ 3’
Allele A2
Allele A1 A G
Note: in a population, individuals will have different combinations of alleles at locus A, e.g. A1A1, A2A2, A1A2, A2A5, A3A6, A4A4, etc
Subject the chromosomes to restriction enzyme digestion/cleavage using an enzyme that cleaves around the SNP (Note: in this example, the enzyme will cleave only allele A2 due to the presence of a G residue, as indicated by an arrow)
A1A1
A1A2
A2A2
900 bp
550 bp
350 bp
What are the expected genotypes?
Dr. Manish Dev Sharma, Assistant professor, Department of Life Sciences, SGRRITS, Dehradun

7. Restriction Fragment Length Polymorphism (RFLP)
The first technology to enable the detection of polymorphism at the DNA sequence level
Based on DNA fragment length differences after digesting genomic DNA with one or more restriction enzymes and separated on an agarose gel (Southern blot procedure is followed, see Lecture 4 notes)
RFLP is able to detect only large shifts in DNA fragments, thus, it can detect insertions and deletions of large sizes and the gain or loss of restriction sites
unable to detect the vast majority of point mutations and insertions or deletions involving small-sized segments because of its low resolution using agarose gel electrophoresis (perhaps consider PAGE!!!)
RFLPs are not necessarily associated with specific genes, therefore the technique was used in genetic mapping studies, for example, to map all the genes in the human genome
Dr. Manish Dev Sharma, Assistant professor, Department of Life Sciences, SGRRITS, Dehradun

8. Molecular basis of RFLP
Deletion causes decreased fragment size
Insertion causes increased fragment size G A
Base changes lead to elimination of sites G A
Base changes lead to gaining of new sites
Chromosome rearrangements
Note: each arrow indicates a restriction site
Dr. Manish Dev Sharma, Assistant professor, Department of Life Sciences, SGRRITS, Dehradun

9. RFLPs display codominant inheritance
8 kb
3 kb
5 kb
Allele A
Allele B
Note: there is an internal restriction site for an enzyme only in allele B
Genotypes
Fragments AA AB BB
8 kb
3 kb, 5 kb, 8 kb
3 kb, 5 kb AA AB BB
8 kb
5 kb
3 kb
Dr. Manish Dev Sharma, Assistant professor, Department of Life Sciences, SGRRITS, Dehradun

10. Consider: cut (+) and no cut (-)
RFLP Mapping - Example
2 kb
7 kb
3 kb
RFLP Locus
Note: the restriction enzyme used always cuts the RFLP locus at the external sites but may or may not cleave the internal sites (individual differences)
Consider: cut (+) and no cut (-)
+/+
+/-
-/-
-/+
2 kb
3 kb
7 kb
10 kb
5 kb
12 kb D B C A
Dr. Manish Dev Sharma, Assistant professor, Department of Life Sciences, SGRRITS, Dehradun

11. RFLP Mapping - Example AB AD CD AC BD BC CC A B C D
Note: consider that a certain (disease-causing) locus has been identified and its inheritance is being studied in a specific family A B C D
Note: circles indicate females and boxes indicate males
Dr. Manish Dev Sharma, Assistant professor, Department of Life Sciences, SGRRITS, Dehradun

12. Random Amplified Polymorphic DNA (RAPD)
RAPD (pronounced “rapid”) is a PCR-based molecular marker technique
it uses single short oligonucleotide primers (ca. 8 – 10 bp) that are arbitrarily selected (i.e. random) to amplify a set of DNA fragments distributed randomly throughout the genome
note: DNA amplification product is generated from a region that is flanked by a part of 10-bp priming sites binding close together and in opposite directions
Genomic DNA from two different individuals often produce different amplification patterns (hence, random amplified polymorphic DNA)
A particular fragment generated for one individual but not the other represents DNA polymorphism and can be used as a genetic marker – “genomic fingerprint”
A DNA sequence difference between individuals in a primer-binding site may result in the failure of the primer to bind, hence no amplification
RAPDs display a dominant pattern of inheritance, i.e. the presence of a band indicates a dominant allele (e.g. AA or Aa) whereas the absence of a band indicates a recessive allele (e.g. aa)
Dr. Manish Dev Sharma, Assistant professor, Department of Life Sciences, SGRRITS, Dehradun

13. RAPD
For each RAPD polymorphism (i.e. phenotype), let us call the “allele” capable for amplification the plus (+) allele and the allele not capable of amplification the minus (-) allele
Three possible genotypes, thus, +/+; +/-; and -/-
This means the homozygous (+/+) and heterozygous (+/-) will both support amplification, whereas the homozygous type (-/-) will not support amplification
Hence, the presence of the amplified fragment is the phenotype observed in both +/+ and +/- (with the + allele being dominant over the - allele)
Note: a specific band occurring at a specific location in all individuals (A – D) in a specific primer set is referred to as being monomorphic
Dr. Manish Dev Sharma, Assistant professor, Department of Life Sciences, SGRRITS, Dehradun

14. Amplified Fragment Length Polymorphism (AFLP)
AFLP is a PCR-based molecular marker technique
essentially a combination of RFLP and RAPD methods
based on the PCR amplification of genomic restriction fragments generated by specific restriction enzymes and oligonucleotide adapters of few nucleotide bases
it’s a DNA fingerprinting technique because it involves the display of a set of DNA fragments from a specific DNA sample
fingerprints are produced without prior sequence knowledge, using a limited set of genetic primers
AFLP involves
genomic DNA digested with res. enzymes (generally two enzymes are used, i.e. one rare cutter such as MseI and another a frequent cutter, e.g. EcoRI)
ligate double-stranded oligonucleotide adapters to the ends of the DNA fragments
(selective) amplification of restriction fragments using radioactively labelled primers. Note: primers are complementary to adapters and may contain one or more extra nucleotides at their 3’-end
gel analysis of the amplified fragments using highly resolving sequencing gels and visualized using autoradiography
Dr. Manish Dev Sharma, Assistant professor, Department of Life Sciences, SGRRITS, Dehradun

15. Restriction enzyme digestion
AFLP Procedure
Genomic DNA
Restriction enzyme digestion
MseI
EcoRI
MseI adapter
EcoRI adapter
Ligate adapters to the ends of restriction fragments
Note: reduces the complexity of restriction ligation products 16-fold
MseI adapter primer
EcoRI adapter primer
Preselective amplification
Dr. Manish Dev Sharma, Assistant professor, Department of Life Sciences, SGRRITS, Dehradun

16. AFLP Procedure - Continued
(MseI primer + 1)
(EcoRI primer + 1) A C
Note: reduces the complexity of PCR product mixture by 256-fold
Selective amplification C A
(MseI primer + 2)
(EcoRI primer + 2) AC CA
Repeat selective amplification using (radioactively/fluorescently-labeled) primers with +2 nucleotides
Polyacrylamide Gel Electrophoresis (PAGE) & Data analysis
Dr. Manish Dev Sharma, Assistant professor, Department of Life Sciences, SGRRITS, Dehradun

17. AFLP Gel Electrophoresis - Example
Different experimental units, e.g. individuals
Monomorphic
Polymorphic
Dr. Manish Dev Sharma, Assistant professor, Department of Life Sciences, SGRRITS, Dehradun

18. Tandem Repeat Polymorphisms
Type of DNA polymorphisms resulting from differences in the number of copies of a DNA sequence that may be repeated many times in tandem at a particular site in a chromosome
any chromosome may have any number of tandem repeats
the number of copies may range from 10 to a few hundred
Note: duplex DNA containing the repeats can also be amplified by means of PCR
Dr. Manish Dev Sharma, Assistant professor, Department of Life Sciences, SGRRITS, Dehradun

19. Simple Sequence Repeats (SSR)
Allele 1
Allele 2
Note: typical SSR has a repeat length of 2 – 9 nucleotides
Dr. Manish Dev Sharma, Assistant professor, Department of Life Sciences, SGRRITS, Dehradun

20. Variable Number Tandem Repeats
Note: due to the larger repeating unit found in VNTRs, they can easily be resolved by electrophoresis such as in DNA typing (or DNA fingerprinting)
Note: unlike SSRs, VNTRs have much longer repeating units, i.e. 10 – 60 nucleotides
Dr. Manish Dev Sharma, Assistant professor, Department of Life Sciences, SGRRITS, Dehradun

21. Molecular Marker Techniques - comparison
Characteristic
RAPD
RFLP
AFLP
SSRs (Microsatellites)
Principle of ID
DNA amplification
Restriction digestion
Principle of Analysis
DNA staining
Southern blotting
Primer requirement
Yes (random)
None
Yes (selective)
Probe requirement
Set of specific probes
Use of radioisotopes No
Yes
Yes/No
Dominant /Codominant
Dominant
Codominant
Dominant/
Polymorphism Detected
Medium
High
Reliability
Intermediate
Dr. Manish Dev Sharma, Assistant professor, Department of Life Sciences, SGRRITS, Dehradun

22. Take Home Message
Genetic markers are based on the presence of DNA sequence variation between individuals within a population or between populations
For a genetic marker to be classified as a good marker system, it must meet several criteria (seldom this is the case)
For this reason, there are different types of genetic marker systems or techniques (e.g. SNPs, RFLPs, RAPDs, AFLPs, SSRs, VNTRs, etc) each with its own advantages and disadvantages
The choice of marker system to employ in a project will depend on several factors including resources available, funds, ease of application, the aims and objectives of the project
Dr. Manish Dev Sharma, Assistant professor, Department of Life Sciences, SGRRITS, Dehradun

23. THANK YOU
Dr. Manish Dev Sharma, Assistant professor, Department of Life Sciences, SGRRITS, Dehradun