MOLECULAR GENETIC MARKERS AND THEIR APPLICATIONS

AnBT : 807 Genome Analysis in Farm Animals

Submitted with respect to : Dr. P. G. Koringa. Assistant Professor,

Submitted by: R. C. Parikh

Ph.D. scholar

Dept. of Animal Biotechnology Veterinary college, AAU, Anand

WHAT IS A MARKER?
Any stable and inherited variation, detectable or measurable by suitable method and which can be used to detect the presence of specific genotype or phenotype that otherwise is not measurable or difficult to detect. The markers could be: MORPHOLOGICAL MARKER: Classical markers CYTOGENETIC MARKERS: Chromosomal aberrations

BIOCHEMICAL MARKERS: Variation in macro-molecules

MORPHOLOGICAL MARKERS

Morphological markers generally correspond to the qualitative traits that can be scored visually. They have been found in nature or as the result of mutagenesis experiments. Morphological markers are usually dominant or recessive

The limitations of morphological markers:

1. 2. 3.

They cause such large effects on phenotype that they are undesirable in breeding programs They mask the effects of linked minor gene(s) They are highly influenced by the environment.

BIOCHEMICAL MARKERS

Isozymes have been used very successfully as biochemical markers in certain plant breeding experiments. Biochemical markers are proteins produced by gene expression . These proteins can be isolated and identified by electrophoresis. They are the products of various alleles of one or several genes.

The limitations of morphological and biochemical markers:

Low polymorphism Requires expression of trait / gene Dominance effect Expression sex limited Expressed late in life

 MOLECULAR (GENETIC) MARKERS:

A molecular marker is a DNA sequence that is readily detected and whose inheritance can easily be monitored . The DNA markers to be utilized may be fragments produced by restriction digestion (like RFLP) or through PCR amplification. Thus they may be hybridization based or PCR based.

Detect variation at DNA level

Does not require expression Not influenced by environment Co-dominant markers, heterozygous can be distinguished Measured in both the sexes at any age e.g. RFLP, PCR-RFLP, RAPD, Minisatellites (VNTRs), AFLP, Microsatellites (STRs/SSRs)

All molecular genetic markers fall under three basic categories: Insertion-Deletion Length Polymorphisms Single Nucleotide Polymorphisms Simple Sequence Repeat Length Polymorphisms (Miniand Micro-Sattellites) Direct Sequencing: The Gold Standard The gold standard in DNA polymorphism analysis is direct sequencing. Direct sequencing uncovers all of the polymorphisms in a DNA target. Because direct sequencing of individuals is obviously impractical on a large scale, numerous methods have been invented to assay for DNA polymorphisms.

RFLP

In 1980 Botstein et al. described the first polymorphic assay. RF stands for Restriction Fragments - fragments of DNA that are cut by restriction enzymes. L stands for Length of the restriction fragments. P stands for Polymorphism (many shapes). The lengths of some of the restriction fragments differ greatly between individuals, thus there are many lengths of DNA possible

Restriction enzymes

Restriction enzymes are isolated from a wide variety of bacterial genera These enzymes are named by using the first letter of the genus, the first two letters of the species, and the order of discovery Restriction endonucleases are enzymes that cleave DNA molecules at specific nucleotide sequences depending on the particular enzyme used. Enzyme recognition sites are usually 4 to 6 base pairs in length. Generally, the shorter the recognition sequence, the greater the number of fragments generated If molecules differ in nucleotide sequence, fragments of different sizes may be generated. The fragments can be separated by gel electrophoresis.

Enzyme EcoR I HpaI Msp I

Microbial origin E.coli H.parainfluenzae Morexella spp.

Restriction site 5..GAATTC..3 3..CTTAA,G..5 5..GTTAAC..3 3..CAAT,TG..5 5..CC GG..3 3..GG C,C..3

Sal I

Streptomycets albus

5..GTC GAC..3 3..CAG CTG..5

RFLP is a sequence of DNA that has a restriction site on each end with a "target" sequence in between for particular RE For example: RE -EcoRI

PCR-RFLP

PCR RFLP
PCR: Exponential amplification of desired segment of DNA in vitro i.e. DNA xeroxing Karry Mullis et al (1986) DNA segment bracketed by two primers amplified by polymerase enzyme using dNTPs in cyclic manner

Simple and fast (duration from days to hours)

Automation Eliminates Southern blotting and autoradiography PCR combined with RFLP Single locus detection

Method

Usually, DNA from an individual specimen is first extracted and purified. Sepatated segment of DNA amplified by PCR.

The DNA is then chopped into restriction fragments by endonucleases which only cut where there are specific DNA sequences recognized by the enzymes. The restriction fragments are then separated according to length by agarose gel electrophoresis

PCR-RFLP Protocol
Reaction mixture for PCR
a. b. c. d. e. f. g. h.

Buffer (10X) MgCl2 (50mM) dNTP (10mM) Primer 1(8pmol) Primer 2(8pmol) DNA template(30ng) Distilled water (sterile) Total

2.5ul 0.75ul 0.5ul 1.0ul 1.0ul 3.0ul 16.5ul 25.0ul

Cycle conditions
1) 2) 3) 4) 5) 6) Initial denaturation 940 C 5min Denaturation 940 C 1min Annealing 55-650 C 1min Extension 720 C 1min Repeat steps 24-29 times Final extension 10min

Reaction Mixture For Restriction Digestion

Buffer(10X) PCR product REs(10Units/ul) Dist. Water Reaction volume Incubate at 370C for 1 hr

3ul 10ul 0.5ul 16.5ul 30ul

The pattern of PCR-RFLPs generated will depends on :

1.Difference in DNA sequence at a particular position between different strains/species 2.The restriction enzyme used.

Difference between conventional RFLP & PCR-RFLP

CONVENTIONAL RFLP 1.Whole genomic DNA 2.Probe is used for identification of particular DNA 3.RE enzymes used before separating particular DNA segment
4.Southern bloting is req. 5.Radioactive probe is req.

PCR-RFLP 1.Particular segment of DNA 2.Primer is used for amplification of particular DNA 3.RE enzymes used after separating particular DNA segment 4.Southern bloting is not req. 5.Radioactive probe is not req.

APPLICATIONS
PCR-RFLPs have provided valuable information in many areas of biology, including: Can be used in paternity cases or criminal cases to determine the source of a DNA sample Can be used determine the disease status of an individual. Can be used to construct genetic map Typing of Microbial Organisms Species identification in meat products using mitochondrial DNA Species determination using PCR RFLP of ancient DNA from Prehistoric Skeletal Remains

Random Amplified Polymorphic DNA (PCR- RAPD)

Williams etal. (1990) developed Random Amplified Polymorphic DNA (RAPD) a technique using very short 10 base primers to generate random fragments from template DNAs Extensively used for genetic characterization in organisms (Bacteria to Mammals) DNA sequences which are relatively variable between different individuals or species. These variable stretches of DNA within a genome among individuals will be useful for analytical method to distinguish individuals from one-another

Utilizes short primers (9-10 bases), usually single to amplify DNA

The particular advantage of this approach of RAPD is that by pooling DNAs from populations of interest Average sets of products obtained in amplification process will mask the variability and highlight more consistent differences between pools/populations

Components of PCR and RAPD Reactions

PCR 1. Buffer (containing Mg++) RAPD 1. Buffer (containing Mg++) - usually high Mg++ concentrations are used lowering annealing stringency 2. Template DNA 3. 1 short primer (10 bases) not known to anneal to any specific part of the template DNA 4. dNTPs 5. Taq DNA Polymerase (or another thermally stable DNA polymerase)

2. Template DNA 3. 2 Primers that flank the

fragment of DNA to be amplified 4. dNTPs 5. Taq DNA Polymerase (or another thermally stable DNA polymerase)

Modifying Thermal Cycling

Two modifications made to typical thermal cycling when RAPD is being done:

1. 2.

3.

Annealing temperatures are generally very low, around 37oC - This allows very short primers to anneal to template DNA A singe 10-mer primer is used instead of a pair of bracketing primers in the conventional/ classical PCR More thermal cycles are used, typically 45 - This compensates for the inefficiency which results from using such short primers.

RAPD

Template DNA

Primer binds to many locations on the template DNA

Only when primer binding sites are close and oriented in
opposite direction so the primers point toward each other, amplification take place

RAPD
Template DNA

Primers point away from each other, so amplification wont happen

RAPD

Template DNA

Primers point in the same direction, so amplification wont happen

RAPD

Template DNA

> 2,000 bases

Primers too far apart, so amplification wont happen

Template DNA

100 - 1,500 bases

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

Separated RAPD Fragments

RAPD reactions were run in groups of 3 using the same template and primer, but varying Magnesium, polymerase and primer concentrations
4mM MgCl2 4mM MgCl2 2mM MgCl2 1.2 U Taq 0.6 U Taq 1.2 U Taq 5 pM OPA-16 10 pM OPA-16 10 pM OPA-16

2 3 4

9 10

Which variable has the greatest impact on fragment patterns? Lowering Magnesium ion concentration results in loss of the largest fragment visible in lanes 2-7

Normal concentrations are shown in yellow text. M = A size standard

RAPD protocol
Reaction mixture Buffer (10x) MgCl2 (50mM) dNTPs(10mM) Primers(8pmol/ul) DNA template(30ng/ul) Distilled water (sterile) 2.5ul 0.75ul 0.5ul 1.0ul 3.0ul 17.25ul

Cycle conditions
Pre denaturation 940C 4min Denaturation 940C 1min Annealing 370C 1min Extension 720C 2min Repeat steps 2-4 for 35-45 times Final extension 720C 5min

Electrophoresis
Electrophoresis conditions required are Matrix:1.5-2% Agarose Buffer:1x TBE Voltage:80volts for 30 min, Visualize under UV light

ADVANTAGES 1) The level of detectable polymorphisms very high 2) In comparison to RFLP, It is less expensive, faster, requires smaller amount of DNA, requires less skill 3) Does not involve use of radioisotopes

DISADVANTAGES 1) Lack of reproducibility 2) PCR results are very sensitive to amplification conditions and consequently variable between laboratories and even between assays

AFLP

METHODOLOGY
1) Digestion of total cellular DNA with one or more restriction enzymes and ligation of restriction half-site specific adaptors to all restriction fragments. 2) Selective amplification of some of these fragments with two PCR primers that have corresponding adaptor and restriction site specific sequences. 3) Electrophoretic separation of amplicons on a gel matrix, followed by visualisation of the band pattern.

Digestion

EcoRI (rare cutter)

MseI (frequent cutter)

Interstitial cohesive ends

Digestion

Adaptor

Adaptor-Ligation

Digestion

Adaptor-Ligation

PCR-Amplification

ADVANTAGES

Extremely sensitive, oligonucleotides used as adaptors and primers. High reproducibility with exactly the same banding patterns being detected over a million-fold difference in initial cDNA concentration. Discriminates heterozygotes from homozygotes when gel scanner used. Allow specific co-amplification of a high number of restriction fragments (50-100in each AFLP reaction) resulting in large number of loci scored per reaction. AFLP are quantitative, in that heterozygote and homozygote genotypes can be differentiated by the intensity of the amplified bands. Choosing the different base number and composition of nucleotides in the adaptors can control the number of DNA fragments that are amplified.

DISADVANTAGES

Highly expensive and requires more DNA than is needed in RAPD (1 mg per reaction). Technically more demanding than RAPDs, as it requires experience of sequencing gels. Relatively complex and requires high-quality DNA; However new software programmes are available to automate scanning and analysis of gels.

APPLICATIONS
Used to distinguish strains. These fingerprints may be used as a tool for determining the identity of a specific DNA samples or to assess the relatedness between samples. Fingerprints are also used as the source for genetic markers to generate linkage maps or to identify molecular markers linked to phenotypic traits and/or genetic loci. AFLP have potentiality in statutory testing for distinguishing new cultivars.

MICROSATELLITES

MICROSATELLITES
Another class of hyper variable loci consisting of short tendem repeats of 2-6 bp.

Also known as Short Tendem Repeats (STR) (Schafer et al., 1988)

Found in every eukaryotic genome but display species specific occurrence and distribution Can be used across the species Highly locus specific marker

Repeat units of Microsatellite markers are flanked by unique sequence. Hence, can be amplified by PCR
Highly polymorphic

PCR based technique: Easy to detect radio active or non radio active

Also called as Simple sequence repeats (SSR) OR Short tandem repeats (STR) For example, AAAAAAAAAAA would be referred to as (A)11 GTGTGTGTGTGT would be referred to as (GT)6 CTGCTGCTGCTG would be referred to as (CTG)4 ACTCACTCACTCACTC would be referred to as (ACTC)4

Feature Origin

RFLP Anonymous/ Genic

SSR Anonymous

RAPD Anonymous

AFLP Anonymous

Isozymes Genic
Limited by the number of enzyme genes and histochemical enzyme assays available (3050)

Maximum theoretical number of possible loci in analysis

Limited by the restriction site (nucleotide) polymorphism (tens of thousands)

Limited by the size of genome and number of simple repeats in a genome (tens of thousands)

Limited by the size of genome, and by nucleotide polymorphism (tens of thousands)

Limited by the restriction site (nucleotide) polymorphism (tens of thousands)

Dominanc e Transferab ility Reproduci bility

Codominant

Codominant

Dominant

Dominant

Codominant Across families and genera

Across genera High to very high

Within genus or species

Within species

Within species Medium to high

Medium to high

Low to medium

Very high

Amount of sample required per sample

2-10 mg DNA

10-20 ng DNA

2-10 ng DNA

0.2-1 g DNA
Moderate to difficult Possible Very good Very limited

Several mg of tissue

Ease of assay Automation / multiplexing Genome and QTL mapping potential Comparative mapping potential Candidate gene mapping potential Potential for studying adaptive genetic variation

Difficult

Easy to moderate Possible Good

Easy to moderate Possible Very good

Easy to moderate Difficult Limited

Difficult Good

Good

Limited

Very limited

Excellent

Limited

Useless

Useless

Useless

Limited

Limited

Limited

Limited

Limited

Good