4. genetic markers

7/31/2019 4. Genetic Markers

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Lecture 3

Types of genetic markers (Lecture in detail) i.e.,

Characteristics and genotyping (Semi-automated and

automated), apparatus used in genotyping.


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The mammalian genome contains of the order of 30,000

genes (even the most recent estimates of gene numberare very controversial, ranging from 30,000 to > 50,000)

Characterstics features of Type I markers:

The final product of a gene is usually a protein,

sometimes an RNA (including ribozymes)

Gene expression can be regulated at the genomic,

transcriptional, post-transcriptional, translational andpost-translational levels.

The mammalian genome contains large numbers of

non-functional genes: processed and unprocessed

pseudogenes, as well as gene fragments.

Type I markers


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Most mammalian genes are "split"

Split genes are made of exons (present in the

mRNA which comprises a leader sequence, a coding

sequence and a trailor sequence) separated by

introns (intervening sequences)

Exons jointly represent approximately 3% of the

genome
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Figure showing: Comparison of a bacterial gene with aeucaryotic gene (SPLIT-GENES).

The bacterial gene consists of a single stretch of

uninterrupted nucleotide sequence that encodes theamino acid sequence of a protein. In contrast, the coding

sequences of most eucaryotic genes (exons) are

interrupted by noncoding sequences (introns). Promoters

for transcription are indicated in green.


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Virtually all genes belong to "gene families"

comprising structurally related genes reflecting a

common evolutionary origin, therefore,

Members of a gene family can be identical in

different species (= "redundant genes")

Members of a gene family can be closely related

(i.e., structural similarity at the nucleotide level)

Members of a gene family can be distantly related

(structural similarity at the protein level)

Members of a gene family can Share evolutionary

related "protein domains" only (possibly by means

of reflecting "exon shuffling")
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Figure showing ORIGIN of GENE FAMILIES: Evolution of thebeta-globin gene family in animals.

An ancestral globin gene duplicated and gave rise to the beta-globin family

(shown here) as well as other globin genes (the alpha family). (A molecule ofhemoglobin is formed from two alpha chains and two beta chains.) The schemeshown was worked out from a comparison of beta-globin genes from manydifferent organisms. For example, the nucleotide sequences of the gammaG andgammaA genes are much more similar to each other than either of them is to theadult beta gene.


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Figure Exon Shuffling: Some results of exon shuffling.

Each type of symbol represents a different family of protein domain, and

these have been joined together end-to-end, as shown, to create larger

proteins, which are identified by name.

T II k


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Tandem repeats = sequences composed of a sequence motif repeated n-times in ahead-to-tail arrangement (Type of tandem repeat)

SatellitesRepeat unit: 10 - > 1,000 bp; repeat length: > 100,000 bpSatellite sequences are primarily concentrated around or at centromeresConstitutive heterochromatin is primarily composed of satellite sequences

TelomeresRepeat unit: 7 bp (GGGGTTA); repeat length:Protect the ends of linear chromosomes

Minisatellites

Repeat unit: 10-100 bp; repeat length: 1000-100,000 bp of size.Thousands of minisatellites are scattered across the genome but arepreferentially located in sub-telomeric regionsFunction (if any) unknownMinisatellites are used for DNA fingerprinting

Microsatellites (Marker of choice in genetic linkage mapping)

Repeat unit: 1-10 bp; repeat length: and about 10-1000 bp in size.

Tens to hundreds of thousands of microsatellites are uniformly scattered acrossthe genome.Function (if any) unknownMicrosatellites are preferred genetic markers in linkage study.

An example of Expansion of trinucleotide repeats underlies inherited disordersshowing anticipation (1 ).

Type II markers

Interspersed repeats: The majority of mammalian interspersed repeats aretransposable elements, including transposons, retroposons and retrotranscripts.
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Gene (Mapped Cytogenetically)

Following information on web

1. Accession number of Gene

2. Map position

3. BP size of the gene

4. No. of Exons and introns

5. BP size of intron & exon

6. Adjacent markers

7. Polymorphic region within gene

8. Primer sequence for genotyping

9. PCR product length

10.Gene function and cDNA sequence

11.Genomic organisation of the gene

12.Related PubMed references

Markers (Mapped genetically)

Following information on web

1. map position (cM)

2. Heterozygosity of marker

3. Primer sequence

4. PCR product size

5. No. of polymarphic alleles

6. Distances with adjacent markers

Gene mapping Genetic mapping

(Type I markers) (Type II markers)1. Mapping of the genes 1. Mapping of molecular markers

2. Genes that are mapped 2. Markers are mapped by linkage

physically by FISH technic. analysis

Propertiesof genetic markers:


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1. Properties of genetic markers:Genetic markers are the basic tool for the molecular genetic study.There are three basic properties of a genetic marker:

locus-specificHighly polymorphic in studying the population (Population genetics)easily genotyped

The quality of a genetic marker is typically measured by its: % Heterozygosity in the population of interest.

PIC (Botstein et al., 1980):Polymorphism Information Content is defined as

The probability that one could identify which homologue of a given

parent was transmitted to a given offspring, the other parent

being genotyped as well.

PIC value = probability that the parent is heterozygous into(x)

probability that the offspring is informative .


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a. Phenotypic Markers:

Phenotypes are the characters for which the variation observed in the

population of interest is entirely explained by a single "mendelian" factor.

Examples:

The seven phenotypes utilized by Mendel ( )The Polled / Horned phenotype in cattleCoat colour variation

Figure 2-3: The seven character differences studied by Mendel
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b. Blood Groups genetic markers

Examples:

Human Blood Groups

Bovine Blood Groups (1)

Porcine Blood Groups (1)

Equine Blood Groups (1)
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c. RFLPs = Restriction Fragment Length PolymorphismsRFLP defined as the observed variation in the restriction map of a given locus

Restrcition enzymes:

Restriction endonucleases cut DNA molecules at specific sites ( )

Recognition sequences for the majority of Type II restriction endonucleases are

palindromes, usually 4-8 bp long ( )

RFLPs can result from:

Point mutation creating or destroying a restriction site ( )

Insertion / deletions altering the length of a given restriction fragment.

RFLPs are usually detected by Southern blotting ( )

A seminal paper based on RFLPs has been the start point of a new era in

mammalian genetics (Botstein et al., 1980).

R t iti R t i ti d l t DNA l l t ifi it ( )
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Restrcition enzymes: Restriction endonucleases cut DNA molecules at specific sites ( )

Figure 10-2: The nucleotide sequences recognized and cut by five widely used restrictionnucleases. As shown, the target sites at which these enzymes cut have a nucleotide sequence andlength that depend on the enzyme. Target sequences are often palindromic (that is, the nucleotidesequence is symmetrical around a central point). In these examples, both strands of DNA are cut atspecific points within the target sequence. Some enzymes, such as Hae III and Alu I, cut straight acrossthe DNA double helix and leave two blunt-ended DNA molecules; for others, such as Eco RI, Not I, and

Hind III, the cuts on each strand are staggered. Restriction nucleases are usually obtained from bacteria,and their names reflect their origins: for example, the enzyme Eco RI comes from Escherichia coli.
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RFLP typing of Sickle cell anemia (B-globulin gene)


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RFLP bySouthern blotting


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e. Minisatellites or VNTR Markers

In 1980, Wyman and White describe the first polymorphism due to allelic

variation in the number of tandem repeats of a minisatellite ( ). They

call these sequences VNTRs (Variable Number of Tandem Repeats).

VNTR markers are usually genotyped using Southern blotting using

restriction enzymes cutting in the sequences flanking the VNTR ( )

In 1985, Jeffreys demonstrates that minisatellites are organized in families

of related sequences and uses this property to develop DNA

fingerprinting systems ( )

VNTR markers and DNA fingerprints have been used extensively for

linkage analysis because of their high informativeness (heterozygosities >

70% are common) but suffer from their uneven genomic distribution.
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Minisatellite of Variable number of tandem repeat (VNTR) markers


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VNTR typing by southern blotting:

VNTR markers are usually genotyped using Southern blotting using

restriction enzymes cutting in the sequences flanking the VNTR

DNA fi i ti b th bl tti


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DNA fingure-printing by southern blotting:

i.e., typing of many VNTR loci as figure-print of an individuals


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DNA figure-print for forensic study:

i.e., Use of DNA figure-print in identification of suspected criminal.

f Microsatellites or SSR Markers
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f. Microsatellites or SSR Markers

In 1989, Weber & May and Litt & Luty discover microsatellite

sequences, demonstrate their high level of

polymorphism due to variations in the number of

tandem repeats ( - typical heterozygosities in cattle),

abundance and even distribution across the genome.

Microsatellites are genotyped using the polymerase chain

reaction ( ) using primers targeted to the unique

sequences flanking the microsatellite motif. PCR can

easily be semi-automated ( )

The resulting PCR products are separated according to size

by gel electrophoresis using either agarose gels or

more commonly (because of their higher resolution)

denaturing polyacrylamide gels (PAGE) ( ).

PCR products are visualized by:

Fig Tri nucleotide repeat microsatellite marker:
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Fig. Tri-nucleotide repeat microsatellite marker:Expansion of the CGG triplet in the FMR-1 gene seen in the fragile Xsyndrome. Normal individuals have from 6 to 54 copies of the CGGrepeat, while individuals from susceptible families display an increase

(premutation) in the number of repeats: normally transmitting males(NTMs) and their daughters are phenotypically normal but display 50 to

200 copies of the CGG triplet; the number of repeats expands to some 200to 1300 in individuals showing full symptoms of the disease.


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PCR for semi-automated Microsatellite

genotyping by auto-radiography.

D t t d P l l id l l t h i


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Denaturated Poly-acrylamide gel electrophoresis

(PAGE) for microsatellite genotyping

Direct staining (ethidium bromide or silverstaining)(1 )
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Direct staining (ethidium bromide or silverstaining)(1)

Autoradiography:

The PCR products are labelled either by incorporation of[a -P 32 or 33] dNTPs (1)or [a -S 35] dNTS during the PCR

amplification (1)(labels the two strands), or by using oneend-labeled primer (labels one strand). Primers are end-labeled using [g -P 32 ]ATP & T4 polynucleotide kinase (1)

After gel electrophoresis, an X-ray film is exposed to the

gel revealing the position of the PCR products as blackspots. A photon of light or a b particle or g ray released froma radioactive molecule "activate" silver bromide crystals onthe film emulsion. This renders them capable of beingreduced through the developing process to form silvermetal (a "grain"). The silver grains on the film form theimage.

Co-amplification and/or co-loading of multiplesmicrosatellites allows for multiplex genotyping (2-4

systems).
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Agarose gelElectrophoresis

(A), (B), (C).

Staining by ethidium

Bromide (B),

Stained by

auto-radiograph (C).

L b li f ti k


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Labelling of dNTP by P32 radio-isotops in microsatellite tying by auto-radiogragy.

Labeling of genetic markers


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Production of PCR product with labeled P32


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Mode of action of P32 labeling in PCR product.

Fluorescence labelling:


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Fluorescence labelling:

The PCR products are labeled either by using primers or dNTPswhich are tagged with an appropriate fluorophore, a chemical groupwhich fluoresces when exposed to a specific wavelength of light.Popular fluorophores used in direct labeling include fluorescein, a

pale green fluorescent dye, rhodamine, a red fluorescent dye, andamino methyl coumarin, a blue fluorescent dye. Fluorophoes arecharacterized by their excitation and emission spectra.

The PCR products are detected during migration using automaticsequencers. ( )

Co-amplification and/or co-loading of multiples microsatellites allowsfor multiplex genotyping (up to 20 systems). ( )

Software packages allow for semi-automated data capture ( ).Microsatellite profiles are often difficult to read due to artefactualbands, which result from (1 ):

the differential migration of the two DNA strands in denaturingacrylamide gelsthe "+ A" activity of the Taq polymerase generating x + 1 bands

slippage of the Taq polymerase during polymerization generating

+4, +2, -2, -4, "stutter" bands

non denatured secondary structures adopted by the PCR products.

Microsatellite typing by automated DNA sequencer:
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Microsatellite typing by automated DNA sequencer:New era of molecular genetics: Using fluorescent labeled primers.(4Pictures of

ABI377 PerkinElmer DNA sequencers)


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g.SNPs (Single nucleotide polymorphisms)The difficulty to fully automate microsatellite genotyping has

revived interest in a new type of markers: single nucleotide

polymorphisms or SNPs.

Definition:

SNPs are polymorphisms due to single nucleotidesubstitutions (transitions > transversions) or single nucleotideinsertions/deletions.

Abundance:The average heterozygosity per nucleotide site, p, has been

estimated at approximately 1/1000 in man, 1/2500 in cattle.

Informativeness:

SNPs are virtually always biallelic markers. Their

heterozygosity is therefore limited at 50%.

Examples of SNP genotyping methods:


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Examples of SNP genotyping methods:1) Single Stranded Conformation Polymorphism (SSCP)( )

2) Allele specific oligonucleotides (ASO)( )

3) Single nucleotide polymorphic discrimination by an electronic dotblot assay (ASO) on semconductor microchips ( ; )4) Reverse dot blot on DNA chips ( )

5) Dynamic allele specific hybridisation (DASH) ( ; )

6) Allele-specific PCR (=amplification refractory mutation system orARMS test)( )

7) Mutation detection the ARMS test in combination with the

TaqmanTM 5' exonuclease assay (exploiting the 5'->3' exonucleaseactivity of Taq DNA polymerase).( )8) Minisequencing and analysis of the extension products by PAGE.

9) Minisequencing and analysis of the extension products on DNAchips

10) Minisequencing and analysis of the extension products using

matrix-assisted laser desorption/ionization time-of-flight massspectrometry (MALDITOF)( )

11) Pyrosequencing ( )

12) OLA ( )

13) Invasive clivage of oligonucleotide probes (Invader technology)

) Si l S d d C f i P l hi (SSCP)(
http://cgil.uoguelph.ca/QTL/SSCP/sld001.htmhttp://cgil.uoguelph.ca/QTL/ASO.htmhttp://cgil.uoguelph.ca/QTL/ECHIP1_files/echip1.htmhttp://cgil.uoguelph.ca/QTL/ECHIP2_files/echip2.htmhttp://cgil.uoguelph.ca/QTL/DNA%20chips/sld001.htmhttp://cgil.uoguelph.ca/QTL/DASH1.htmhttp://cgil.uoguelph.ca/QTL/DASH2A.htmhttp://cgil.uoguelph.ca/QTL/ARMSTest.htmhttp://cgil.uoguelph.ca/QTL/Taqman.htmhttp://cgil.uoguelph.ca/QTL/MALDI1.htmhttp://cgil.uoguelph.ca/QTL/Pyro1.htmhttp://cgil.uoguelph.ca/QTL/latifa/sld001.htmhttp://cgil.uoguelph.ca/QTL/INV1.htmhttp://cgil.uoguelph.ca/QTL/INV1.htmhttp://cgil.uoguelph.ca/QTL/latifa/sld001.htmhttp://cgil.uoguelph.ca/QTL/latifa/sld001.htmhttp://cgil.uoguelph.ca/QTL/Pyro1.htmhttp://cgil.uoguelph.ca/QTL/Pyro1.htmhttp://cgil.uoguelph.ca/QTL/MALDI1.htmhttp://cgil.uoguelph.ca/QTL/MALDI1.htmhttp://cgil.uoguelph.ca/QTL/Taqman.htmhttp://cgil.uoguelph.ca/QTL/Taqman.htmhttp://cgil.uoguelph.ca/QTL/ARMSTest.htmhttp://cgil.uoguelph.ca/QTL/ARMSTest.htmhttp://cgil.uoguelph.ca/QTL/DASH2A.htmhttp://cgil.uoguelph.ca/QTL/DASH2A.htmhttp://cgil.uoguelph.ca/QTL/DASH1.htmhttp://cgil.uoguelph.ca/QTL/DASH1.htmhttp://cgil.uoguelph.ca/QTL/DNA%20chips/sld001.htmhttp://cgil.uoguelph.ca/QTL/DNA%20chips/sld001.htmhttp://cgil.uoguelph.ca/QTL/ECHIP2_files/echip2.htmhttp://cgil.uoguelph.ca/QTL/ECHIP2_files/echip2.htmhttp://cgil.uoguelph.ca/QTL/ECHIP1_files/echip1.htmhttp://cgil.uoguelph.ca/QTL/ECHIP1_files/echip1.htmhttp://cgil.uoguelph.ca/QTL/ASO.htmhttp://cgil.uoguelph.ca/QTL/ASO.htmhttp://cgil.uoguelph.ca/QTL/SSCP/sld001.htmhttp://cgil.uoguelph.ca/QTL/SSCP/sld001.htmhttp://cgil.uoguelph.ca/QTL/SSCP/sld001.htm


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1) Single Stranded Conformation Polymorphism (SSCP)((Figure 1-3)
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Detection and production of Allele specific Oligo-nucleotide (ASO)


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Detection and production of Allele specific Oligo-nucleotide (ASO)

Allele-specific oligonucleotide (ASO) dot-blot hybridisation can identify individuals with the sickle cell mutation.The sickle cell mutation is a single nucleotide substitution (A to T) at codon 6 in the b -globin gene, resulting in aGAG (Glu) to GTG (Val) substitution. The example shows how one can design ASOs: one specific for the normal(b A) allele and identical to a sequence of 19 nucleotides encompassing codons 3-9 of this allele, and one specificfor the mutant (b S) allele, being identical to the equivalent sequence of the mutant allele. The labeled ASOs canbe individually hybridized to denatured genomic DNA samples on dot-blots. The b A- and b S-specific ASOs canhybridize to the complementary antisense strand of the normal and mutatnt alleles respectively, forming perfect

19-bp duplexes. However, duplexes between the b A-specific ASO and the b S allele, or between the b S-specificASO and the b A allele have a single mismatch and are unstable at high hybridization stringency.

2) Single nucleotide polymorphic discrimination by an


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2) Single nucleotide polymorphic discrimination by anelectronic dot blot assay (ASO) on semiconductor microchip

Single nucleotide polymorphic discrimination by an electronic


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S g e uc eot de po y o p c d sc at o by a e ect o cdot blot assay (ASO) on semiconductor microchips

Correct base-pairing at the 3' end of PCR primers is the basis of allele-specific


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p g p pPCR.

The allele-specific oligonucleotide primers ASP1 and ASP2 are designed to be identical tothe sequence of the two alleles over a region preceding the position of the variantnucleotide, up to and terminating in the variant nucleotide itself. ASP1 will bind perfectly tothe complementary strand of the allele 1 sequence permitting amplification with conserved

primer. However, the 3'-terminal C of the ASP2 primer mismatches with the T of the allele 1sequence, making amplification impossible. Similarly ASP2 can bind perfectly to allele 2 andinitiate amplification, unlike ASP1.

Pyrosequencing is


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sequencing by synthesis. ' A simpleto use technique for accurate andconsistent analysis of large numbersof short to medium length DNAsequences.

Step1.A sequencing primer is hybridized toa single stranded, PCR amplified,DNA template, and incubated withthe enzymes, DNA polymerase, ATPsulfurylase, luciferase and apyrase,and the substrates, adenosine 5

phosphosulfate (APS) and luciferin.

4. genetic markers

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