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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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