MOLECULAR MARKERS

Key issues 1. 2. 3. 4. 5. Introduction (some definitions) Marker-assisted biotechnology Molecular structures targeted for diagnostic and evolutionary work Features that render them suitable Techniques used for parasite diagnostic / evolutionary work (theory and procedure)

Introduction Markers are heritable variable traits that indicate the underlying genetic differences between individuals, populations, species or higher taxa. They include Biological, Morphological, Biochemical, Cytological and DNA-based or Molecular markers Molecular marker General definition: Molecular Markers are specific fragments of DNA that can be identified within the whole genome. They are based on naturally occurring polymorphisms in DNA sequences (i.e. base pair deletions, substitutions, additions or patterns). Molecular markers are found at specific locations of the genome. Genetic markers for example are used to 'flag' the position of a particular gene or the inheritance of a particular characteristic. Coding regions are less variable than non-coding regions presumably due to the selection against changes that render proteins non functional than the original version. In genetics, a molecular marker (identified as genetic marker) is a fragment of DNA sequence that is associated to a part of the genome OR it is a gene or DNA sequence with a known location on a chromosome that can be used to identify cells, individuals or species specifically. It can be described as a variation (which may arise due to mutation or alteration in the genomic loci) that can be observed. A genetic marker may be a short DNA sequence, such as a sequence surrounding a single base-pair change (single nucleotide polymorphism, SNP), or a long one, like minisatellites. Molecular markers are used in molecular biology and biotechnology experiments to identify a particular sequence of DNA. Since the DNA sequences are very highly specific, they can be identified with the help of the known molecular markers which can find out a particular sequence of DNA from a group of unknown. Markers can be further categorized as dominant or co-dominant. Dominant markers allow for analyzing many loci at one time, e.g. RAPD. A primer amplifying a dominant marker could amplify at many loci in one sample of DNA with one PCR reaction. Co-dominant markers analyze one

locus at a time. A primer amplifying a co-dominant marker would yield one targeted product. Co-dominant markers (RFLPs, microsatellites, etc) allow the analysis of only a locus per experiment, so they are more informative because the allelic variations of that locus can be distinguished Marker-assisted biotechnology Marker assisted selection or marker aided selection (MAS) This is a process whereby a marker (morphological, biochemical or one based on DNA/RNA variation) is used for indirect selection of a genetic determinant or determinants of a trait of interest (i.e. productivity, disease resistance, abiotic stress tolerance, and/or quality). The trait of interest is selected not based on the trait itself but on a marker linked to it (1, 2). For example if MAS is being used to select individuals with a disease, the level of disease is not quantified but rather a marker allele which is linked with disease is used to determine disease presence. The assumption is that linked allele associates with the gene and/or quantitative trait locus (QTL) of interest. MAS can be useful for traits that are difficult to measure, exhibit low heritability, and/or are expressed late in development. It is applied in animal and plant breeding. Using DNA-based and/or molecular markers, a unique DNA sequence, occurring in proximity to the gene or locus of interest, can be identified by a range of molecular techniques such as RFLPs, RAPDs, AFLP, DAF, SCARs, microsatellites etc. Molecular structures targeted for diagnostic and evolutionary work 1. ITS (Internal Transcribed Spacer): Piece of non-functional RNA situated between structural ribosomal RNAs (rRNA) on a common precursor transcript in eukaryotic organisms such as Trypanasomes. Eukaryotic ribosomal RNA genes (known as ribosomal DNA or rDNA) are found as parts of repeat units that are arranged in tandem arrays, located at the chromosomal sites known as nucleolar organizing regions (NORs). Each repeat unit consists of a transcribed region (having genes for 18S, 5.8S and 26S rRNAs and the external transcribed spacers i.e. ETS1 and ETS2) and a non-transcribed spacer (NTS) region. In the transcribed region, internal transcribed spacers (ITS) are found on either side of 5.8S rRNA gene and are described as ITS1 and ITS2.The length and sequences of ITS regions of rDNA repeats are believed to be fast evolving and therefore may vary. Universal PCR primers designed from highly conserved regions flanking the ITS and its relatively small size (600-700 bp) enable easy amplification of ITS region due to high copy number (up to-30000 per cell, Dubouzet and Shinoda 1999) of rDNA repeats. This makes the ITS region an interesting subject for evolutionary/phylogenetic investigations (Baldwin et al. 1995; Hershkovitz et.al. 1996, 1999) as well

as biogeographic investigations (Baldwin 1993; Suh et al. 1993; Hsiao et al. 1994; Dubouzet and Shinoda 1999). ITS region has also been studied to assess genetic diversity as it evolves very quickly. E.g the differences in the ribosomal RNA genes (18SSU) at species and strain levels are a basis of specific diagnostic procedures for accurate identification of Cryptosporidium parasites in clinical and environmental samples. ITS1 is useful in P. falciparum studies. 2. Tandem repeats Occur in DNA when a pattern of two or more nucleotides is repeated and the repetitions are directly adjacent to each other. Example: A-T-T-C-G-A-T-T-C-G-A-T-T-C-G, the sequence A-T-T-C-G is repeated three times. When the number of repeating nucleotides is not known, variable, or irrelevant, it is sometimes called a variable number tandem repeat (VNTR).
Variable Number Tandem Repeat (or VNTR)

It is a location in a genome where a short nucleotide sequence is organized as a tandem repeat. These can be found on many chromosomes, and often show variations in length between individuals. Each variant acts as an inherited allele, allowing them to be used for personal or parental identification. Their analysis is useful in genetics and biology research, forensics, and DNA fingerprinting. The repeats are tandem - they are clustered together and oriented in the same direction. Individual repeats can be removed from (or added to) the VNTR via recombination or replication errors, leading to alleles with different numbers of repeats. Flanking the repeats are segments of nonrepetitive sequence (shown here as thin lines), allowing the VNTR blocks to be extracted with restriction enzymes and analyzed by RFLP, or amplified by the polymerase chain reaction (PCR) technique and their size determined by gel electrophoresis whe they produce a pattern of bands unique to each individual. VNTRs with very short repeat blocks may be unstable - dinucleotide repeats may vary from one tissue to another within an individual, while trinucleotide repeats have been found to vary from one generation to another (see Huntington's disease) There are two minisatellites. (a) Mini satellites Minisatelite defined as regions of DNA with polymorphisms in the number of repeated nucleotide sequences of approximately 25bp in length, found in the euchromatic regions of the genome of vertebrates, fungi and plants. 10 to 60 nucleotides are repeated. Difference in number of minisatelites is the basis for DNA finger printing. principal families of VNTRs: microsatellites and

(b)Microsatellites These are repeats of sequences less than about 5 base pairs in length. The terms Short Tandem Repeat (STR) and Simple Sequence Repeat (SSR) which are more descriptive are also used to mean microsatellites. Used to examine genetic variation in Cryptosporidium spicies. Microsatelites are found in vertebrates, insects and plant genomes. Are abundant in P. falciparum genome.

1. Single Nucleotide Polymorphisms (SNPs) SNP is a DNA sequence variation occurring when a single nucleotide in the genome differs between members of a biological species or paired chromosomes in an individual. Alternatively, Single Nucleotide Polymorphisms can be defined as a single base change in a DNA sequence with a usual alternative of two possible nucleotides at a given position e.g. P. falciparum multi drug resistance gene pfmdr-1. SNPs are the most common genetic polymorphisms and they occur at an appreciable frequency in the genome. They are distributed throughout genome with high density SNP are more stable and easy to assay SNPs are detected using specific primers to amplify alleles of interest Used to identify allelic specific polymorphisms

SNPs identification possible using; (a) SSCP- Single Strand Conformation Polymorphism This is based on difference in patterns of DNA folding due to single nucleotide changes. Mobility of such DNA strands differs in the non denaturing gel electrophoresis (b) Heteroduplex analysis It is performed by identifying the patterns of mobility of homo and heteroduplexed in non denaturing gel electrophoresis or HPLC Advantage Possible to identify mutations due to single base substitutions.
1. Conserved Sequences

Present in all members of the same species.

2. Introns: Non coding sequences in genes absent in Tryps 3. Spacers: Refers to a piece of non-functional RNA situated between

structural ribosomal RNAs (rRNA) on a common precursor transcript.

4. Pallindromic sequences: Restriction sites are usually palindrome. 5. Transposons: These are mobile genetic elements

Features that render the above molecular structures suitable targets Highly polymorphic nature Codominant inheritance (determination of homozygous and heterozygous states of diploid organisms) Frequent occurrence in genome Selective neutral behaviour (the DNA sequences of any organism are neutral to environmental conditions or management practices) Easy access (availability) Easy and fast assay High reproducibility Easy exchange of data between laboratories.

Markers generated by the specific techniques 1. Restriction Fragment Length Polymorphism (RFLP) This is a technique that exploits variations in homologous DNA sequences. It refers to a difference between samples of homologous DNA molecules that come from differing locations of restriction enzyme sites, and to a related laboratory technique by which these segments can be illustrated. May simply refer to gain or loss of restriction sites associated with a genetic locus. Mutations can result in loss of restriction sites to generate different sized DNA fragments at a given locus. The technique involves digestion of Genomic DNA with enzymes to yield restriction fragments. Fragments are separated with agarose gel electrophoresis and detected by Southern blotting using appropriate probes. Fragments identified by presence or absence of restriction sites

Restriction sites are often palindromes Used to asses genetic diversity of parasites DNA finger printing Advantage

RFLP markers are co-dorminant (allows determination of homozygous and heterozygous states of diploid organisms) Disadvantages Require large amount of DNA samples

The method is also laborious and takes several days

1. Amplified Fragment Length Polymorphism (AFLP) It aims at generating few random fragments which can be compared between organisms. It uses 2 restriction enzymes; one 6 cutter e.g. Hind III and one 4 cutter e.g. Taq I followed by ligation of adaptors of known to sticky ends of restriction fragments. A subset of the restriction fragments to be amplified is then selected using labelled primers complementary to the adaptor sequence, the restriction site sequence and a few nucleotides inside the restriction site fragments. The amplified fragments are visualized on denaturing polyacrylamide gels either through autoradiography or fluorescence methodologies. The amplified sequences are compared to identify the polymorphic loci. AFLPPCR is a highly sensitive method for detecting polymorphisms in DNA. It is used as a tool to study genetic diversity of protozoa and helminths. Advantages It has higher reproducibility, resolution, and sensitivity can amplify between 50 and 100 fragments at one time. No prior sequence information is needed for amplification (Meudt & Clarke 2007). Beneficial in the study of taxa of various organisms. Disadvantage Typically dominant markers 1. Random Amplified Polymorphism (RAPD)

RAPD markers based on a PCR reaction in which the segments of DNA amplified are random. Short PCR primers (approx 10 bases) are used arbitrarily to hybridize along with DNA fragments at low annealing temps Fragments hybrised at sufficient intervals along DNA selectively are amplified Changes within such regions prevent primer binding and essentially amplification. Only fragments complementary to primers get amplified.This achieves selective amplification of DNA sequences with RAPD markers. RAPD often shows a dominant relationship due to primers being unable to bind on recessive alleles RAPD products are usually visualized on agarose gels stained with ethidium bromide.

It is used to examine strain level genetic variation in a variety of parasites e.g leishmania, nematodes, and toxoplasma. RAPD: Randomly amplified polymorphic DNA

1. Cleaved Amplified Polymorphic Sequences

It is a technique in molecular biology for the analysis of genetic markers. It is an extension of RFLP. PCR is used to specifically amplify particular regions of genomic DNA. Selection achieved by use of specific primers. Amplified fragments are then digested endonucleases to reveal polymorphism with restriction

When fractionated by agarose or acrylamide gel electrophoresis, the digested PCR products will give readily distinguishable patterns of bands. Alternatively, the amplified segment can by analyzed by Allele specific oligonucleotide (ASO) probes usually by a simple Dot blot. Advantages CAPS markers codominant hence easy identification of homo and heterozygous patterns Small amounts of DNA since PCR used Disadvantage Requires specific primers and hence the sequence must already be known

1. Simple sequence length polymorphism

SSLPs are short tandem repeated sequences over varying base lengths (less than 5 base pairs) in intergenic regions of Deoxyribonucleic Acid (DNA). They are used as genetic markers with Polymerase Chain Reaction (PCR). An SSLP is a type of polymorphism: a difference in DNA sequence amongst individuals. Variance in the length of SSLPs can be used to understand genetic variance between two individuals in a certain species. Specific primers complementary to regions of microsatellites are used to identify the SSLPs. Hybridisation depends on number of the repeats and so do the amplified fragment size. Agarose gel electrophoresis used to separate polymorphic fragments Used to examine strain level genetic variation in cryptosporidium and toxoplasma. Doesnt employ restriction enzymes hence avoids problems of partial digestion Co dominant

Advantages

Diagnosis of parasitic infections

Direct microscopy is widely used for the diagnosis of parasitic infections although it often requires an experienced microscopist for accurate diagnosis, is labour intensive and not very sensitive. To overcome some of these shortcomings, molecular or nucleic acid-based diagnostic methods for parasitic infections have been developed. Early methods, which involved hybridisation of specific probes (radiolabelled and non-

radiolabelled) to target deoxyribonucleic acid (DNA), have been replaced by more sensitive polymerase chain reaction (PCR)-based assays. Other methods, such as PCR-hybridisation assays, PCR-restriction fragment length polymorphism (PCR-RFLP) assays and random amplified polymorphic DNA (RAPD) analysis are also valuable for epidemiological studies of parasites (International journal of parasitology, volume 27, 1997). (A)Molecular techniques 1. Polymerase chain reaction It is a scientific technique in molecular biology used to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. In diagnosis, PCR is used to amplify a specific region of a DNA strand (the DNA target) unique to a pathogen or known group of organisms as the basis for detection. Amplified fragments usually range from 10 to 40 kilo bases. The process is rapid and simple, yielding a large number of copies from very small amounts of DNA, even when the source is relatively of poor quality. Requirements include: Template: At least 1 intact DNA strand of the organism being investigated should be present. Any impurities are sufficiently diluted (1:5 dilution optimal) Primers: short segments of DNA that provide specifity Deoxynucleoside triphosphates (dNTPs) namely; dATP, dTTP, dCTP, dGTP which are the building-blocks from which the DNA polymerase synthesizes a new DNA strand. Thermostable DNA polymerase (e.g., Taq, Pfu) Buffer and salts (KCl, MgCl2) which provide a suitable chemical environment for optimum activity and stability of the DNA polymerase. Optional: BSA, DMSO, Formamide

Repeated cycles of melting (strand separation), primer annealing, and primer extension by cycling temperatures

A PCR cycle theoretically doubles the amplicon

To check whether the PCR generated the anticipated DNA fragment, agarose gel electrophoresis is used for size separation of the PCR products. The size(s) of PCR products is determined by comparison with a DNA ladder (a molecular weight marker), which contains DNA fragments of known size, run on the gel alongside the PCR products. During diagnosis and evolutionary studies of parasites, PCR amplifies target sequences & increases such as; (a) Ribosomal DNA/RNA: provide high sensitivity but are not good for closely related species. (b) Specific sequences of genomic DNA: Highly specific for single species not sensitive. (c) Random primer amplification (RADP) PCR: Very highly sensitive - not specific. Examples: PCR diagnosis of Eimeria protozoa involves amplification of a 500 base pair sequence in ribosomal RNA requires 1pg DNA - < 10 oocysts. Cryptosporidium parvum protozoa diagnosis involves amplification of approximately a 400 base pair sequence from faeces. It is sensitivity to 6 oocysts per g faeces.

C. parvum specimen

positive

faecal

Other PCRbased molecular techniques for diagnostic and evolutionary studies of parasites 1. Single strand conformation polymorphism analysis (SSCP) PCR Principle: single-stranded DNAs with single base substitutions migrate at different speeds.

Primers are labelled or labelled deoxyribonucleotide in the used dNTP. It detects single nucleotide changes quickly. 2. Restriction Fragment Length Polymorphism (RFLP) DNA is digested with restriction enzymes of choice and gel electrophoresis is performed to separate fragments. The DNA is transferred to a nitrocellulose filter where it is hybridised with labelled c DNA probe for the selected sequence or gene. The nitrocellulose is then autoradiographed to see bands. 3. PCR-RFLP Technique PCR amplifies a short region of DNA having restriction sites. The amplified DNA with restriction sites is digested with appropriate restriction enzymes and gel electrophoresis performed to separate fragments in agarose containing ethidium bromide. The gel is visualised under UV to detect and analyse presence of restriction sites and band profiles which are unique to the organism. E.g. two bands indicate presence of one restriction site while one band indicates absence of restriction sites. 4. Allele sequence- specific oligonucleotide hybridisation It is used in discrimination of amplification products that differ by a single base pair. Amplified DNA is immobilised on nylon membrane, labelled sequencespecific oligonucleotide is used to identify DNA products with mutant or normal sequences. DNA-RNA hybrids with mismatches digested with RNAse. 5. DNA Microarray techniques DNA chips are commercially prepared. Oligonucleotides are spotted on specific positions on a glass and hybridised with fluorescent labelled test DNA. Test DNA or RNA hybridises to oligonucleotide that matches its sequence. Chips are read and data analysed by a computer. 6. Southern Blotting Electrophoretically separated DNA IS transferred from a gel to a solid support (such as nylon or nitrocellulose membrane) and probed with oligonucleotides that are specific for the particular sequence of DNA nucleotides. Example; Southern blot patterns of Eimeria protozoa using ribsosomal RNA probes Requires micrograms of DNA. Advantages of Nucleic acid based molecular diagnosis

 Genomic DNA constant -parasite & hosts contain unique DNA sequences . Very sensitive - small biopsy. Probes can be designed with flexibility:

Specific - detect single parasite species. Less specific - detect group of parasites.
Disadvantages: Expensive - especially PCR. Radioactivity needed: newer non-radioactive probes. PCR can fail: - Contamination & false positives. DNA probes do not distinguish between dead & living parasites (A)Biochemical tests: enzyme patterns Isoenzymes Perform same functions but have different movement on gels, a factor which is genetically controlled. That is, parasites with different gel patterns for isoenzymes are genetically distinct. Isoenzymes can be separated by charge (Isoelectric focusing ) or size (SDS-PAGE) Example: Isoenzyme Analysis of Chagas Disease

Advantage of Assessment of enzyme pattern based diagnosis Simple technique. Large number of typing enzymes available. Many samples typed at same time. Power to distinguish morphologically similar parasites. Disadvantages: Significant tissue needed for analysis e.g visceral leishmaniasis requires spleen, liver. Technique not rapid can take days. Sometimes incorrect diagnosis enzyme labile.

 Technique simple but equipment expensive. (A)Immunological tests Rely on identification of specific antibodies or antigens. 1. Enzyme-Linked Immunosorbant Assay (ELISA) for diagnosis. In Direct ELISA, the antigen or antibody is attached to a solid phase and reacted with a enzyme-labelled antibody or antigen respectively. In Indirect ELISA, the antigen is attached to a solid phase and serum containing antibodies (primary antibody) to the antigen is added. A secondary antibody (against the primary antibody ) conjugated to an enzyme is then added. Enzyme substrate is added to cause a colour reaction. 2. CATT (Card Agglutination Test for Trypanosomiasis) For diagnosis of African sleeping sickness. Used for simple and rapid detection of Anti-trypanosomal IgM Drop of blood Mixed with fixed parasites on plastic card Blue granular deposits = infection Advantages of immunological techniques Rapid easy field-based tests. Both individual & mass population screening. Ig subclasses to improve specificity / sensitivity. Disadvantages: Some cannot distinguish past / present infections. Cannot distinguish morphologically similar parasites. Expensive to develop significant Research prior to commercialization Other immunological assays Chagas Disease 1. FATALA kit : measures T. cruzi antibodies recombinant proteins in blood using 2

2. BIO CHAGAS kit: uses cocktail recombinant T. cruzi antigens. Infection sera produce blue precipitate on strip in 60 minutes. (A)Microscopic techniques Examples: Blood smears for haemoparasites such as malaria parasites and trypanosomes, skin snips for Onchocerciasis, faecal smear / urine

filtration for Schistosomiasis, lumbar puncture for Human African Trypanasomiasis (HAT), Cryptosporidium parvum protozoa faecal analysis (limit 50,000 oocysts per gram of faeces).e.t.c REFERENCES Baldwin BG, Sanderson MJ, Porter JM, Wojciechowski MF, Campbell CS and Donoghue MJ 1995 The ITS region of nuclear ribosomal DNA: a valuable source of evidence on angiosperm phylogeny. Ann. Mo. Bot. Gard. 82: 247-277.Baldwin et al. 1995; Hershkovitz et.al. 1996, 1999 Baldwin BG 1993 Molecular phylogenetics of Calcydenia (Compositae) based on ITS sequences of nuclear ribosomal DNA: Chromosomal and morphological evolution reexamined. Am. J. Bot. 80(2): 222-238. Hershkovitz MA and Zimmer EA 1996 Conservation patterns in angiosperm rDNA ITS2 sequences. Nucleic Acid Research. 24: 2857-2867. Hershkovitz MA, Zimmer EA and Hahn WJ 1999 Ribosomal DNA sequences and angiosperm systematics. In: P.M. Hollingsworth, R.M. Bateman and R.J. Gornall eds. Molecular systematics and plant evolution. Taylor & Francis, London pp.268-326. Dubouzet JG and Shinoda K 1999 Relationships among old and New world Alliums according to ITS DNA sequence analysis. Theor. Appl. Genet. 98: 422-433.
Jo Hamilton Parasitology BS31820

Meudt HM, Clarke AC (March 2007). "Almost forgotten or latest practice? AFLP applications, analyses and advances" Ribaut, J.-M. and Hoisington, D. A., Marker assisted selection: new tools and strategies. Trends Plant Sci., 1998, 3, 236239. Ribaut, J.-M. et al., , 2001 Genetic basis of physiological traits. In Application of Physiology in Wheat Breeding, CIMMYT, Mexico. Rosyara, U.R. 2006. REQUIREMENT OF ROBUST MOLECULAR MARKER TECHNOLOGY FOR PLANT BREEDING APPLICATIONS.Journal of Plant Breed.