methods for characterization of animal genomes(snp,str,qtl,rflp,rapd)

Methods for characterization of animal genomes (RFLP, RAPD,

SNP, STR, QTL )

Submitted by: Dr. Vijayata

Restriction Fragment Length Polymorphism (RFLP)

• RFLP is a technique in which organisms may be differentiated

by analysis of patterns derived from cleavage of their DNA.

• If two organisms differ in the distance between sites of

cleavage of particular Restriction Endonucleases, the length of

the fragments produced will differ when the DNA is digested

with a restriction enzyme.

Potential of Molecular Markers in Plant Biotechnology, P. Kumar1&2, V.K. Gupta2, A.K. Misra2, D. R. Modi*1 and B. K. Pandey2 Plant Omics Journal 2(4):141-162 (2009) ISSN: 1836-3644

• The similarity of the patterns generated can be used to

differentiate species (and even strains) from one another.

• This technique is mainly based on the special class of enzyme

i.e. Restriction Endonucleases.

• They have their origin in the DNA rearrangements that occur

due to evolutionary processes, point mutations within the

restriction enzyme recognition site sequences, insertions or

deletions within the fragments, and unequal crossing over

(Schlotterer & Tautz, 1992).


• This is not always the case with genomic DNA molecules

because some restriction sites are polymorphic, existing as two

alleles, one allele displaying the correct sequence for the

restriction site and therefore being cut when the DNA is treated

with the enzyme, and the second allele having a sequence

alteration so the restriction site is no longer recognized.

• The result of the sequence alteration is that the two adjacent

restriction fragments remain linked together after treatment with

the enzyme, leading to a length polymorphism .

Genomes, 2nd edition Terence A BROWN Chapter 5. Mapping Genomes, 5.2.2. DNA markers for genetic mapping. Oxford: Wiley-Liss; 2002.ISBN-10: 0-471-25046-5

The DNA molecule on the left has a polymorphic restriction site (marked with the asterisk) that is not present in the molecule on the right. The RFLP is revealed after treatment with the restriction enzyme because one of the molecules is cut into four fragments whereas the other is cut into three fragments.

A restriction fragment length polymorphism (RFLP)


• In order to score an RFLP, it is necessary to determine the size of

just one or two individual restriction fragments against a

background of many irrelevant fragments.

• Size fractionation is achieved by gel electrophoresis and, after

transfer to a membrane by Southern blotting; fragments of interest

are identified by hybridization with radioactive labeled probe.

• Different sizes or lengths of restriction fragments are typically

produced when different individuals are tested.


• Southern hybridization, using a probe that spans the

polymorphic restriction site, provides one way of visualizing

the RFLP , but nowadays PCR is more frequently used.

• The primers for the PCR are designed so that they anneal

either side of the polymorphic site, and the RFLP is typed by

treating the amplified fragment with the restriction enzyme

and then running a sample in an agarose gel .


(A) RFLPs can be scored by Southern hybridization. The DNA is digested with the appropriate restriction enzyme and separated in an agarose gel. The smear of restriction fragments is transferred to a nylon membrane and probed with a piece of DNA that spans the polymorphic restriction site. If the site is absent then a single restriction fragment is detected (lane 2); if the site is present then two fragments are detected (lane 3).

Two methods for scoring an RFLP :


(B) The RFLP can also be typed by PCR, using primers that anneal either side of the polymorphic restriction site. After the PCR, the products are treated with the appropriate restriction enzyme and then analyzed by agarose gel electrophoresis. If the site is absent then one band is seen on the agarose gel; if the site is present then two bands are seen.


Applications:

• RFLPs can be applied in diversity and phylogenetic studies

ranging from individuals within populations or species, to

closely related species.

• RFLPs have been widely used in gene mapping studies

because of their high genomic abundance due to the ample

availability of different restriction enzymes and random

distribution throughout the genome (Neale & Williams 1991).

• RFLP markers were used for the first time in the construction

of genetic maps by Botstein et al.1980.


Random Amplified Polymorphic DNA (RAPD)

• PCR based technology.

• RAPD is a DNA polymorphism assay based on the

amplification of random DNA segments with single primers of

arbitrary nucleotide sequence (Williams et al., 1990; Welsh and

McClelland, 1990).


• In this reaction, a single species of primer anneals to the genomic

DNA at two different sites on complementary strands of DNA

template.

• If these priming sites are within an amplifiable range of each other,

a discrete DNA product is formed through thermo cyclic

amplification.

• On an average, each primer directs amplification of several discrete

loci in the genome, making the assay useful for efficient screening

of nucleotide sequence polymorphism between individuals .


• Amplified products are separated on agarose gel (1.5-2.0%)

and visualised by ethidium bromide staining .

• The use of a single oligonucleotide promotes the generation of

several discrete DNA products and these are considered to

originate from different genetic loci.

• Polymorphisms result from mutations or rearrangements either

at or between the primer binding sites and are detected as the

presence or absence of a particular RAPD band .

• This means that RAPDs are dominant markers and, therefore,

can not be used to identify heterozygotes.

Random amplified polymorphic DNA (RAPD) markers and its applicationsN. Senthil Kumar1 and G. Gurusubramanian2 , 2011 Sci Vis 11 (3), 116-124.

• The standard RAPD utilises short synthetic oligonucleotides (10

bases long) of random sequences as primers to amplify nanogram

amounts of total genomic DNA under low annealing temperatures

by PCR.

• Welsh and McClelland independently developed a similar

methodology using primers about 15 nucleotides long and different

amplification and electrophoretic conditions from RAPD and called

it the arbitrarily primed polymerase chain reaction (AP-PCR)

technique.Random amplified polymorphic DNA (RAPD) markers and its applicationsN. Senthil Kumar1 and G. Gurusubramanian2 , 2011 Sci Vis 11 (3), 116-124.

• PCR amplification with primers shorter than 10 nucleotides

[DNA amplification fingerprinting (DAF)] has also been used

to produce more complex DNA fingerprinting profiles.

• Although these approaches are different with respect to the

length of the random primers, amplification conditions and

visualization methods, they all differ from the standard PCR

condition in that only a single oligonucleotide of random

sequence is employed and no prior knowledge of the genome

subjected to analysis is required.Random amplified polymorphic DNA (RAPD) markers and its applicationsN. Senthil Kumar1 and G. Gurusubramanian2 , 2011 Sci Vis 11 (3), 116-124.

Applications:

• The application of RAPDs and their related modified markers

in variability analysis and individual-specific genotyping has

largely been carried out, but is less popular due to problems

such as poor reproducibility faint or fuzzy products, and

difficulty in scoring bands, which lead to inappropriate

inferences.

• RAPDs have been used for many purposes, ranging from

studies at the individual level (e.g. genetic identity) to studies

involving closely related species.


• RAPDs have also been applied in gene mapping studies to fill

gaps not covered by other markers (Williams et al. 1990,

Hadrys et al. 1992).

• Variants of the RAPD technique include Arbitrarily Primed

Polymerase Chain Reaction (AP-PCR), which uses longer

arbitrary primers than RAPDs, and DNA Amplification

Fingerprinting (DAF) that uses shorter, 5–8 bp primers to

generate a larger number of fragments.

• Multiple Arbitrary Amplicon Profiling (MAAP) is the

collective term for techniques using single arbitrary primers.


Microsatellites / Short tandem repeats (STRs)

• The term microsatellites was coined by Litt & Lutty (1989)and

it also known as Simple Sequence Repeats (SSRs).

• Microsatellites, or short tandem repeats/simple sequence

repeats (STRs/SSRs), are polymorphic loci present in DNA

that consist of repeating units of one to six base pairs in length

(CACACACACACACACA) (Litt and Lutty, 1989; Tautz,

1989).

A Brief Review of Molecular Techniques to Assess Plant Diversity,Arif et al.,2010Int. J. Mol. Sci. 2010, 11, 2079-2096; doi:10.3390/ijms11052079

• The repeated sequence is often simple, consisting of two, three

or four nucleotides (di-, tri- and tetra- nucleotide repeats) and

can be repeated many times.

• Microsatellites can be amplified for identification by PCR

using the unique sequences of flanking regions as primers.


• The most common way to detect microsatellites is to design

PCR primers that are unique to one locus in the genome and

that base pair on either side of the repeated portion.

• Therefore, a single pair of PCR primers will work for every

individual in the species and produce different sized products

for each of the different length of microsatellites.

• The PCR products are separated either by slab gel

electrophoresis or capillary gel electrophoresis in an

automated sequencer.


• The number of repeats can be determined by carrying out a

PCR using primers that anneal either side of the STR, and then

examining the size of the resulting product by agarose or

polyacrylamide gel electrophoresis .

GENE CLONING AND DNA ANALYSIS An Introduction T.A. BROWN, Faculty of Life Sciences University of Manchester Manchester, Sixth Edition Part II The Applications of Gene Cloning and DNA Analysis in Research P180

• Another approach using SSRs for the detection of

polymorphism, called inter simple sequence repeat (ISSR),

involves PCR amplification of genomic DNA between two

microsatellite loci using a single primer composed of a

microsatellite sequence anchored at the 3’ or 5’ end with one or

few arbitrary, often degenerate nucleotides.

• These primers target simple sequence repeats that are abundant

throughout the eukaryotic genome and do not require prior

knowledge of DNA sequence for primer design.

Biotechnology & Genetic Engineering Reviews Volume 25, Editor: Stephen E. Harding, First published 2008

• Microsatellites have proved to be versatile molecular markers,

particularly for population analysis, but they are not without

limitations.

• With the abundance of PCR technology, primers that flank

microsatellite loci are simple and quick to use, but the

development of correctly functioning primers is often a

tedious and costly process.


Applications:

• In general, microsatellites show a high level of polymorphism.

• As a consequence, they are very informative markers that can be

used for many population genetics studies, ranging from the

individual level (e.g. clone and strain identification) to that of

closely related species (distinguish closely related genotypes;

because of their high degree of variability).

• Microsatellites are also considered ideal markers in gene

mapping studies .Potential of Molecular Markers in Plant Biotechnology, P. Kumar1&2, V.K. Gupta2, A.K. Misra2, D. R. Modi*1 and B. K. Pandey2 Plant Omics Journal 2(4):141-162 (2009) ISSN: 1836-3644

Single Nucleotide Polymorphism (SNP)

• Single nucleotide polymorphism (SNP) is a DNA sequence

variation occurring when a single nucleotide (A, T, G or C)

differs among members of a species. (Single nucleotide

variations in genome sequence of individuals of a population are

known as SNPs.)


• They are bi-allelic markers, indicating a specific

polymorphism in two alleles only of a population.

• Most SNPs (about two of every three SNPs), involve the

replacement of cytosine (C) with thymine (T).

These are positions in a genome where some individuals have one nucleotide (e.g. a G) and others have a different nucleotide (e.g. a C)


• SNPs represent one of the more interesting approach in

genotypization, because they are abundant in the genome,

genetically stable and amenable to high-throughput automated

analysis (Vignal et al., 2002).

• SNP genotyping technologies have two components -

a method for determining the type of base present at a given

SNP locus , and a method for reporting the presence of the

allele(s) (signal detection).

MOLECULAR MARKERS IN ANIMAL GENOME ANALYSIS, A. TenevaBiotechnology in Animal Husbandry 25 (5-6), p 1267-1284, 2009

• SNP detection is more rapid because it is based on

oligonucleotide hybridization analysis.

• An oligonucleotide is a short single-stranded DNA molecule,

usually less than 50 nucleotides in length, that is synthesized in

the test tube.


• An oligonucleotide will hybridize with another DNA molecule

only if the oligonucleotide forms a completely base-paired

structure with the second molecule.

• If there is a single mismatch - a single position within the

oligonucleotide that does not form a base pair - then

hybridization does not occur .


• Oligonucleotide hybridization can therefore discriminate

between the two alleles of an SNP.

• Various screening strategies have been devised ( Mir and

Southern, 2000), including DNA chip technology and solution

hybridization techniques.

• A DNA chip is a wafer of glass or silicon, 2.0 cm2 or less in

area, carrying many different oligonucleotides in a high-density

array.

• The DNA to be tested is labeled with a fluorescent marker and

pipetted onto the surface of the chip.


• Hybridization is detected by examining the chip with a fluorescence

microscope, the positions at which the fluorescent signal is emitted

indicating which oligonucleotides have hybridized with the test

DNA.

• Many SNPs can therefore be scored in a single experiment (Gerhold

et al., 1999).

• Solution hybridization techniques are carried out in the wells of a

microtiter tray, each well containing a different oligonucleotide, and

use a detection system that can discriminate between unhybridized

single-stranded DNA and the double-stranded product that results

when an oligonucleotide hybridizes to the test DNA.Genomes, 2nd edition Terence A BROWN Chapter 5. Mapping Genomes, 5.2.2. DNA markers for genetic mapping. Oxford: Wiley-Liss; 2002.ISBN-10: 0-471-25046-5

• SNP have become the preferred markers in genetic disease

studies for various livestock species, as researches direct their

attention towards functional genomics (White et al., 2001).

• SNPs are becoming especially important as markers because they

are very stable, i.e. have very low mutation rates and can be

amplified with PCR for testing.

• One disadvantage of these markers is the lower informational

content compared with that of a highly polymorphic

microsatellite, but it can be compensated by the use of a higher

number of markers (Werner et al. 2002, 2004).

MOLECULAR MARKERS IN ANIMAL GENOME ANALYSIS, A. TenevaBiotechnology in Animal Husbandry 25 (5-6), p 1267-1284, 2009

Quantitative trait loci (QTLs)

• The regions within genomes that contain genes associated with a

particular quantitative trait are known as quantitative trait loci

(QTLs).

• The identification of QTLs based only on conventional phenotypic

evaluation is not possible.

• A major breakthrough in the characterization of quantitative traits

that created opportunities to select for QTLs was initiated by the

development of DNA (molecular) markers in the 1980s.An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic concepts , B.C.Y. Collard1,4, , M.Z.Z. Jahufer2, J.B. Brouwer3 & E.C.K. Pang1 , ∗ 2005. Springer 142: 169–196.

QTL mapping

• The process of constructing linkage maps and conducting QTL

analysis to identify genomic regions associated with traits is known

as QTL mapping (also ‘genetic,’ ‘gene’ or ‘genome’ mapping) .

• The most important use for linkage maps is to identify chromosomal

locations containing genes and QTLs associated with traits of

interest; such maps may then be referred to as ‘QTL’ (or ‘genetic’)

maps.

• ‘QTL mapping’ is based on the principle that genes and markers

segregate via chromosome recombination (called crossing-over)

during meiosis (i.e. sexual reproduction), thus allowing their

analysis in the progeny (Paterson, 1996a). An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic concepts , B.C.Y. Collard1,4, , M.Z.Z. Jahufer2, J.B. Brouwer3 & E.C.K. Pang1 , ∗ 2005. Springer 142: 169–196.

• QTL analysis is based on the principle of detecting an association

between phenotype and the genotype of markers.

• Markers are used to partition the mapping population into

different genotypic groups based on the presence or absence of a

particular marker locus and to determine whether significant

differences exist between groups with respect to the trait being

measured .

• A significant difference between phenotypic means of the groups

(either 2 or 3), depending on the marker system and type of

population, indicates that the marker locus being used to partition

the mapping population is linked to a QTL controlling the trait. An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic concepts , B.C.Y. Collard1,4, , M.Z.Z. Jahufer2, J.B. Brouwer3 & E.C.K. Pang1 , ∗ 2005. Springer 142: 169–196.

• Three widely-used methods for detecting QTLs are single-

marker analysis, simple interval mapping and composite

interval mapping (Liu, 1998; Tanksley, 1993).

• Single-marker analysis (single-point analysis) is the

simplest method for detecting QTLs associated with single

markers.

• The statistical methods used for single-marker analysis include

t-tests, analysis of variance (ANOVA) and linear regression.

• This method does not require a complete linkage map and can

be performed with basic statistical software programs.

An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic concepts , B.C.Y. Collard1,4, , M.Z.Z. Jahufer2, J.B. Brouwer3 & E.C.K. Pang1 , ∗ 2005. Springer 142: 169–196.

• However, the major disadvantage with this method is that the

further a QTL is from a marker, the less likely it will be

detected.

• This is because recombination may occur between the marker

and the QTL.

• This causes the magnitude of the effect of a QTL to be

underestimated (Tanksley, 1993).

• The use of a large number of segregating DNA markers

covering the entire genome may minimize both problems

(Tanksley, 1993).


• The simple interval mapping (SIM) method makes use of

linkage maps and analyses intervals between adjacent pairs of

linked markers along chromosomes simultaneously, instead of

analyzing single markers (Lander & Botstein, 1989).

• The use of linked markers for analysis compensates for

recombination between the markers and the QTL, and is

considered statistically more powerful compared to single-

point analysis (Lander & Botstein, 1989; Liu, 1998).


• Composite interval mapping (CIM) has become popular for

mapping QTLs.

• This method combines interval mapping with linear regression

and includes additional genetic markers in the statistical model in

addition to an adjacent pair of linked markers for interval

mapping (Jansen, 1993; Jansen & Stam, 1994).

• The main advantage of CIM is that it is more precise and

effective at mapping QTLs compared to single-point analysis and

interval mapping, especially when linked QTLs are involved.


Type of markers Advantages Disadvantages

Restriction FragmentLength Polymorphism(RFLP)

-High genomic abundance-Co-dominant markers-Highly reproducible-Good genome coverage-Can be used across species-No sequence information-Needed for map based cloning

-Need large amount of good quality DNA-Laborious (compared to RAPD)-Difficult to automate-Need radioactive labeling-Cloning and characterization of probe are required

Randomly AmplifiedPolymorphic DNA(RAPD)

-High genomic abundance-Good genome coverage-No sequence information-Ideal for automation-Less amount of DNA -No radioactive labeling-Relatively faster

-No probe or primer information-Dominant markers-Not reproducible-Can not be used across species-Not very well-tested

Simple SequenceRepeat (SSR)

-High genomic abundance-Highly reproducible-Fairly good genome coverage-High polymorphism-No radioactive labeling-Easy to automate-Multiple alleles

-Can not be used across species-Need sequence information-Not well-tested


Feature RFLP RAPD STR/SSR

Amount of DNA required High Low

Low

Maximum theoreticalnumber of possible loci in analysis

Limited by therestriction site(nucleotide)

Polymorphism

Limited by thesize of genome, and

by nucleotide polymorphism

Limited by the size ofgenome and number of

simple repeats in aGenome

Dominance Codominant Dominant Codominant

Genomic abundance High High Medium

Locus specificity Yes No No

ReproducibilityHigh to very

highLow tomedium

Medium tohigh

Type of probes/primers

Low copy genomic DNA or

cDNA clones

Usually 10 bprandom nucleotides

Specific repeat DNA sequence

MOLECULAR MARKERS IN ANIMAL GENOME ANALYSIS, A. Teneva Biotechnology in Animal Husbandry 25 (5-6), p 1267-1284, 2009

Comparison of commonly used genetic markers (Mburu and Hanotte , 2005)

Feature RFLP RAPD STR/SSR

Degree of Polymorphism

High Low Medium

Type of polymorphism

Single base changes,insertion, deletion

Single base changes,insertion, deletion Changes in length of

repeats

Potential forstudyingadaptive geneticVariation

Limited Limited Limited

TransferabilityAcross relatedspecies

Across genera Within species Within genus or species

Ease of development Difficult Easy Difficult

Technical requirement

High Low Medium

Major application Physical mapping Gene tagging Genetic diversity

MOLECULAR MARKERS IN ANIMAL GENOME ANALYSIS, A. Teneva Biotechnology in Animal Husbandry 25 (5-6), p 1267-1284, 2009

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