PCR-based Molecular MarkersIntroduction:-Background:- Differences that distinguish one organism from others are based on the composition of their genotype. Nucleotide sequences (genes) that control traits can be located/traced by the junk DNA portions or non coding sequences present in the near proximity. By observing these non coding sequences scientist can make genetic linkage map serve for analyzing QTLs, paternity testing and introgression of genes.

Classification of molecular markers.

Genetic markers are of three distinct types:-

1. Visually assessable traits (traits that can be studied morphologically like agronomic traits).2. Biochemical markers (based on gene products like allozymes monoterpenes).3. DNA based genetic markers ( ESTs,VNTR,SSR)

Summary of common molecular marker

SINGAL LOCUS(Co-Dominant) DETECTION

1 ) Restriction Fragment Length Polymorphism (RFLP) Hybridization2) Cleaved amplified polymorphic sequences(CAPS) PCR3) Simple Sequence Length Polymorphism(SSLP) PCRo Variable number of tandem repeates (VNTR) Hybridization

or PCR [using Minisatellites]

o Simple sequence repeats/ PCR Simple tandem repeats (SSR/STP) [using microsatellites]

4) Sequence characterized amplified region (SCAR) PCR5) Single nucleotide polymorphism (SNPs) o Dynamic allele-specific hybridization Hybridizationo DNA chips

Hybridizationo DNA sequencing Sequencingo Single strand conformation polymorphism(SSCP) Conformation

MULTIPLE LOCI (Dominant)

1) Amplified product length polymorphism (AFLP)

2) Random amplified polymorphic DNA (RAPD)

3) DNA amlification fingerprinting (DAF)

4) Arbitrarily primed-PCR (AP-PCR)

5) Simple sequence length polymorphism (SSLP) when multiple pairs of primer were used

6) Inter-simple sequence repeat (ISSR)

7) Single nucleotide polymorphism (SNP) -Single strand conformation polymorphism (SSCP)

when used to scan for random located SNPS

DETECTION

PCR

PCR

PCR

PCR

PCR

PCR

Conformation

Types of Molecular markers techniques.

ASAP Allele Specific associated primers

ASO Allele specific oligos

AS-PCR Allele specific-polymerase chain reaction

AFLP Amplified fragment length polymorphism

AMP-PCR Anchored microsatellite primed-PCR

ASSR Anchored simple sequence repeats

AP-PCR Arbitrary primed-PCR

CAPS Cleaved amplified polymorphic sequence

DOP-PCR Degenerated oligonucleotide primed-PCR

Dart Diversity arrays technology

DAF DNA amplification fingerprinting

EST Expressed sequence tags

ISSR Inter-simple sequence repeats

IPCR Inverse-PCR

ISTR Inverse sequence tags

MP-PCR Microsatellite primed –PCR

MASDA Multiplexed allele-specific diagnostic assay

RAMP Random amplified microsatellite polymorphism

RAM Random amplified microsatellite

RAPD Random amplified polymorphic DNA

SSLP Simple sequence length polymorphism

SSR Simple sequence repeats

SNP Single nucleotide polymorphism

RFLP Restriction fragment length polymorphism

SAMPL Selective amplification of microsatellite polymorphic loci

SCAR Sequence characterized amplified regions

SSAP Sequence specific amplification polymorphism

STMS Sequence tagged microsatellite

STS Sequence tagged site

STR Short tandem repeats

SPAR Single primer amplification repeats

SSCP Single standard conformational polymorphism

SSI Site selected insertion –PCR

SDA Strand displacement amplification

YNTR Variable number tendem repeats

Why we consider Molecular markers?

(1) To locate any variation or mutation in genetic sequence.

(2) To create a genetic linkage map or database.

(3) To study associations between traits of great economic and biotechnological importance and their responsible genes (polygene may be).

(4) To study interspecific traits and interspecific breeds(within a genus) so in result we can observe transfer of genes ,satellites from one gene pool of one species into other species, we can also form interbreed cross on basis of molecular markers assisted breeding. Usually markers are not normal genes ,mostly they are DNA sequence with no hereditable effects, instead they are indexes of genes location.

Characteristics studied by Molecular markers

(1) Mode of inheritance of genes (I) Bi-parental nuclear inheritance(both female & male chromosomal portion(nuclear genes). (ii) Maternal Organelle inheritance and paternal organelle maternal organelle inheritance ( Biparental Organelle inheritance)

(2) Mode of gene action:- Dominant or co-dominant markers. (i) Dominant:- A dominant marker allows amplification of several loci in

one PCR with one sample of DNA( e.g RAPD.AFLP,SAMPLS) (ii) Co-dominant:- Marker analyze one locus at a time ,more informative( RFLP, Microsatellite).because at a time a particul locus allelic variations can be distinguished.

1. Hybridization based (hybridization of DNA segments by hybridizing probes and restriction enzymes) e.g RFLP

2. PCR based techniques. two main types:- (1) Arbitrary primer (without previously known sequence). (2) Known DNA sequences( which are going to be amplified).

Arbitrarily Amplified DNA Markers. MAAP multiple arbitrary amplicons profiling. (Caetan

anoles et al.,)(RAPD or AP-PCR or DAF , ), AFLP,ISSR These techniques are used to differentiate two individuals of a

species.

Site-targeted PCR techniques Develop from known DNA sequences ( e.g EST, CAPS,SSR,SCAR,STS) used to differentiate genotype of two different species.

RFLP Principle:-

Restriction fragment length polymorphism (RFLP) technique was developed in 1980s primarily for mapping human genome and was later applied to plants.

We can generate many different DNA fragments by digesting total DNA with specific restriction enzymes.

These fragments are relatively small in size and are co-dominant in nature i.e both strands can be assayed.

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.

The analysis requires a relatively complex technique that is time consuming and expensive.

RFLP

PRINCIPLE The hybridization results can be visualized by

1. Autoradiography (if the probes are radioactively labeled) 2. Chemiluminesence (if non-radioactive, enzyme-link methods are

used for probe labeling and detection, digoxigenin, antibodies etc). Uses:-

Locating disease causing mutants.Further prediction of inheritance pattern. (which one would be carrier( or homozygous for disease causing allele)

An autoradiograph detecting parent (P1&P2) and homozygous and heterozygous (H ) F1 segregation

PCR-Based Techniques. RAPD (Random Amplification of polymorphic

DNA.)

RAPD also known as,AP-PCR(Arbitrarily primed PCR),DAF (DNA amplification fingerprinting)& MAAP(Multiple arbitrary amplicon profiling)

DNA segment can be amplified using short oligodeoxynucleotide primer of arbitrary nucleotide sequence (amplifier) and polymerase chain reaction procedure (Khal et al., 2001 )

. RAPDs are produce by PCR using genomic DNA and arbitrary primers

Taq polymerase is used to amplify DNA segment between closely spaced sequence (< 2kb) and complementary to the short random oligomers (typically 10-mers)

RAPD polymorphism result from change in the primer-binding site in the DNA sequence

RAPD

RAPD Segments of DNA to be amplified are arbitrary or random.

Short primers mostly 10 nucleotides or 8-12 bp are used. Arbitrary primers should have at least 40% GC content, mostly used 50-80%(Williams et al.,) so that they can withstand annealing temperature i.e. 72 Celsius Absence of palindromic sequence.

PCR products obtained are run on 1.6%-2% agarose gel stained with Ethidium Bromide or with polyacrylamide in combination with radioactive labeled nucleotide.

The band obtained after running on gel shows two types of polymorphism, brighter bands show homozygous dominance and less brighter will hetrozygously dominant. so it is very difficult to distinguish or differentiate between them, so markers are known as Dominant markers, and homozygous recessive DNA fragments or loci are not amplified

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

Template DNA

Primers point in the same direction, so amplification won’t happen

Template DNA

Primers too far apart, so amplification won’t happen

> 2,000 bases

Template DNA

Primers are just the right distance apart, so fragment is amplified

100 - 1,500 bases

Applications

Case Study

To assess the extent of genetic variability that exist and also to establish phylogenetic relationship among the species of Jatropha.

Jatropha curcas,J. glandulifera, J. gossypifolia, J. integerrima,J. multifida, J. podagrica and J. Tanjorensis

using RAPD and AFLP

Jatropha is a genus of flowering plants in the spurge family, Euphorbiaceae. The name is derived from the Greek words ἰατρός, meaning "physician," and τροφή, meaning "nutrition," hence the common name physic nut. Higher classification: EuphorbiaceaeLower classifications: Jatropha curcas,

RAPD profiles of different species of Jatropha L. with primer OPL 5 (1–7) and IDT E 4. (8–14).

Lane 1 & 8 —J. tanjorensis; 2 & 9 —J. curcas; 3 & 10—J. glandulifera; 4 & 11—J. gossypifolia; 5 & 12—J. multifida; 6 & 13—J. podagrica; 7 & 14—J. integerrima;

M—1 Kb DNA ladder

Limitations of RAPD

Low Reproducibility.This issue can be eradicate by proper handling and using efficient protocol without any contaminants

Dominant Marker effectRAPD doesn’t differentiate between Hetero and homozygous dominant alleles, no fragments will be produced from homozygous recessive allele.

Homology Mismatches between the primer and the template may result in the total absence of PCR product as well as in a merely decreased amount of the product. Thus, the RAPD results can be difficult to interpret.

AFLP

AFLP(Amplified fragment length polymorphism)

Difference between corresponding DNA fragment from two organisms can be detected by amplification of restricted fragments, hence polymorphism can be observed.

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 (18-24bp)of known sequence are then ligated 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.

AFLP Adapters for Eco R1 And Mse

Eco :- (1) 5’- CTC GTA GAC TGC GTA CC - 3’Eco :- (2) 5’-AAT TGG TAC GCA GTC TAC-3’Mse:- (1) 5’ GAC GAT GAG TCC TGA G-3’Mse:- (2) 5’ TAC TCA GGA CTC AAT-3’{Whole genomic DNA + Restriction enzymes + adaptors + ligase enzyme}

now Mse 1(5’-3’) arrange to anneal with 5’-3’target strand.and Mse 2(3’-5’) Arrange to anneal with 3’-5’ target strandsimilarly Eco 1 with 5’-3’ target dna strand and Eco 2 with 3’-5’ strand.

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

Principle

By no selective base

PCR Amplification take place either

………AAGTN………TTCAN

NAAGT……..NTTCA……...

………TTCA

AAGT…….By 1, 2, or 3 selective base

………AAGTN………TTCAN

NAAGT……..NTTCA……...

………TTCAA

GAAGT………..Primer(+1) for pre-selective amplification

………AAGTNNN………TTCANNN

NNNAAGT……..NNNTTCA……...TAGAAGT………..

………TTCAACG

Primer(+3) for selective amplification

4 time reduction in amplification16 time reduction in amplification64 time reduction in amplification

Case study

AFLP profiles of different species of Jatropha L. with selective amplification with primers CAT/E-AAG, (1–7); M-CAA/EACA(8–14) andM-CAA/E-ACT (15-21).

Lane 1, 8 & 15 — J. Curcas2, 9 & 16 —J. tanjorensis; 3, 10 & 17 —J. glandulifera;4, 11 & 18 —J. gossypifolia;5, 12 & 19 —J. multifida; 6, 13 & 20 —J. podagrica; 7,14 & 21 —J. integerrima; M—1 Kb + 100 bp DNA ladder mix

Case study

Application of AFLP

Advantage Disadvantage

CAPS

Cleaved amplified polymorphic sequences (CAPS)

They are co-dominant genetic markers that use PCR amplification and subsequent restriction endonuclease digestion to detect [single nucleotide] polymorphisms (SNPs) or insertion/deletion (INDELs) in a region of interest (Konieczny and Ausubel 1993).

When a SNP is found that distinguishes alleles underlying a desired trait, the CAPS marker becomes a functional marker and should be 100% predictive of the presence/absence of the allele and its corresponding phenotype.

Introduction & Principle

CAPS

Introduction & Principle

Variety A Variety B

Mutation occur EcoRI sitePCR amplification and restriction digestion

Case study

The rice xa5 gene for disease resistance to Xanthomonas oryzae encodes the gamma subunit of transcription factor IIA (TFIIAgamma). TFIIAgamma is a general eukaryotic transcription factor with no previously known role in disease resistance. xa5 is unusual in that it is recessive. Sequencing of TFIIAgamma in resistant and susceptible isolines revealed substitutions of two nucleotides(in dominant), which results in an amino acid change from valine to glutamic acid at position 39 of the protein resulting resistant and susceptible cultivars.

Due to the change in 2 nucleotide a restriction site is created in dominant gene and hence with the help of CAPS it is used in MAS

Case study

Case study

Resistance Susceptible

Application of CAPS

Advantage Disadvantage

Application of CAPS

ISSR-PCR

1. ISSR involves amplification of DNA segments present at an amplifiable distance in between two identical microsatellite repeat regions oriented in opposite direction (Figure9). The technique uses microsatellites as primers in a single primer PCR reaction targeting multiple genomic loci to amplify mainly inter simple sequence repeats of different sizes. The microsatellite repeats used as primers' for ISSRs can be di-nucleotide, tri-nucleotide, tetra-nucleotide or penta-nucleotide. The primers used can be either unanchored (Meyer et al., 1993; Gupta et al., 1994;Wu et al., 1994) or more usually anchored at 3` or 5` end with 1 to 4 degenerate bases extended into the flanking sequences ( Zietkiewicz et al., 1994; Figure 9). ISSRs use longer primers (15–30 mers) as compared to RAPD primers (10 mers), which permit the subsequent use of high annealing temperature leading to higher stringency.

2. The annealing temperature depends on the GC content of the primer used and ranges from 45 to 65C. The amplified products are usually 200–2000 bp long and amenable to detection by both agrose and polyacrylamide gel electrophoresis. ISSRs exhibit the specificity of microsatellite markers, but need no sequence information for primer synthesis enjoying the advantage of random markers (Joshi et al., 2000). The primers are not proprietary and can be synthesized by anyone. The technique is simple, quick, and the use of radioactivity is not essential. ISSR markers usually show high polymorphism (Kojima et al., 1998)

Application of ISSR

REMAP & IRAP

Definition

Any difference in DNA sequence between two genomes, detected by polymerase chain reaction-mediated amplification of the region between a long terminal repeat of a retrotransposon and a nearby microsatellite

The dispersion, ubiquity and prevalence of retrotransposon-like elements in plant genomes can be exploited for DNA-fingerprinting

Two DNA techniques based on retrotransposon like elements are introduce here

IRAP (Inter-Retrotransposon Amplified Polymorphism) ,

REMAP (REtrotransposon-Microsatellite Amplified Polymorphism) (Kalendar et al., 1999).

Principle

Principle

The IRAP (Inter- Retrotransposon Amplified Polymorphism) markers are generated by the proximity of two retrotransposons using outward facing primers annealing to their long terminal repeats (LTRs)

Retrotransposon

Principle

In REMAP (Retrotransposon-Microsatellite Amplified Polymorphism) the DNA sequence between the LTRs and adjacent microsatellites (SSRs) are amplified using appropriate primers

Microsatellite Retrotransposon

Application of IRAP & REMAP

SNPs

Definition

Any polymorphism between two genomes that is based on a single nucleotide exchange, small deletion or insertion

Principle

Small nucleotide polymorphism (SNP) is a new marker technology originally developed in human. SNPs are the most abundant polymorphic marker with 2-3 polymorphic sites every kilobase (Cooper et al., 1985). Originally discovered in humans, SNPs have now been developed for genotyping in plants. SNP technology is heavily dependent upon sequence data. Several methods are available for SNP detection including

Automated fluorescent sequencing denaturing high-performance liquid chromatography (DHPLC, Underhill et al., 1996), DNA microarrays (Hacia and Collins, 1999), Single-strand conformational polymorphic-capillary electrophoresis (SSCP-CE, Ren, 2001)Microplate array diagonal-gel electrophoresis (MADGE, Day et al., 1998) and Matrix-assisted laser desorption /ionisation time of flight (MALDI-TOF, Griffin and Smith, 2000)

Principle

Application of SNPs

Advantage Disadvantage