Lecture 3 Types of genetic markers (Lecture in detail) i.e.
, Characteristics and genotyping (Semi-automated and
automated), apparatus used in genotyping.
Type I markers
The mammalian genome contains of the order of 30,000 genes (even the most recent estimates of gene number are 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 and post-translational levels. The mammalian genome contains large numbers of non-functional genes: processed and unprocessed pseudogenes, as well as gene fragments.
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
Figure showing: Comparison of a bacterial gene with a eucaryotic gene (SPLIT-GENES). The bacterial gene consists of a single stretch of uninterrupted nucleotide sequence that encodes the amino 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.
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")
Figure showing ORIGIN of GENE FAMILIES: Evolution of the beta-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 of hemoglobin is formed from two alpha chains and two beta chains.) The scheme shown was worked out from a comparison of beta-globin genes from many different organisms. For example, the nucleotide sequences of the gammaG and gammaA genes are much more similar to each other than either of them is to the adult beta gene.
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.
Type II markers
Tandem repeats = sequences composed of a sequence motif repeated n-times in a
head-to-tail arrangement (Type of tandem repeat) Satellites Repeat unit: 10 - > 1,000 bp; repeat length: > 100,000 bp Satellite sequences are primarily concentrated around or at centromeres Constitutive heterochromatin is primarily composed of satellite sequences Telomeres Repeat 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 are preferentially located in sub-telomeric regions Function (if any) unknown Minisatellites 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 across the genome. Function (if any) unknown Microsatellites are preferred genetic markers in linkage study. An example of Expansion of trinucleotide repeats underlies inherited disorders showing anticipation (1 ).
Interspersed repeats: The majority of mammalian interspersed repeats are
transposable elements, including transposons, retroposons and retrotranscripts.
Properties of genetic markers:
Gene mapping
(Type I markers) 1. Mapping of the genes 2. Genes that are mapped physically by FISH technic.
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
Genetic mapping (Type II markers)
1. Mapping of molecular markers 2. Markers are mapped by linkage analysis 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
Genetic markers Type II (in detail)
1. Properties of genetic markers 2. Type of molecular markers:
a. Phenotypic Markers b. Blood Groups c. Biochemical Polymorphisms d. RFLPs
e. Minisatellites or VNTR Markers
f. Microsatellites or SSR Markers g. SNPs
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-specific Highly 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 .
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 (1 ) The Polled / Horned phenotype in cattle Coat colour variation
Figure 2-3: The seven character differences studied by Mendel
b. Blood Groups genetic markers
Examples: Human Blood Groups
Bovine Blood Groups (1)
Porcine Blood Groups (1) Equine Blood Groups (1)
c. RFLPs = Restriction Fragment Length Polymorphisms
RFLP defined as the observed variation in the restriction map of a given locus Restrcition enzymes: Restriction endonucleases cut DNA molecules at specific sites (1 )
Recognition sequences for the majority of Type II restriction endonucleases are
palindromes, usually 4-8 bp long (1 )
RFLPs can result from: Point mutation creating or destroying a restriction site (1 ) Insertion / deletions altering the length of a given restriction fragment. RFLPs are usually detected by Southern blotting (1 ) A seminal paper based on RFLPs has been the start point of a new era in mammalian genetics (Botstein et al., 1980).
Restrcition enzymes: Restriction endonucleases cut DNA molecules at specific sites (1 )
Figure 10-2: The nucleotide sequences recognized and cut by five widely used restriction nucleases. As shown, the target sites at which these enzymes cut have a nucleotide sequence and length that depend on the enzyme. Target sequences are often palindromic (that is, the nucleotide sequence is symmetrical around a central point). In these examples, both strands of DNA are cut at specific points within the target sequence. Some enzymes, such as Hae III and Alu I, cut straight across the 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.
RFLP typing of Sickle cell anemia (B-globulin gene)
RFLP by Southern blotting
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 (1 ). 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 (1 )
In 1985, Jeffreys demonstrates that minisatellites are organized in families of related sequences and uses this property to develop DNA fingerprinting systems (1 1 ) 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.
Minisatellite of Variable number of tandem repeat (VNTR) markers
VNTR typing by southern blotting:
VNTR markers are usually genotyped using Southern blotting using
restriction enzymes cutting in the sequences flanking the VNTR
DNA fingure-printing by southern blotting: i.e., typing of many VNTR loci as figure-print of an individuals
DNA figure-print for forensic study:
i.e., Use of DNA figure-print in identification of suspected criminal.
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 (1 - typical heterozygosities in cattle), abundance and even distribution across the genome. Microsatellites are genotyped using the polymerase chain reaction (1 ) using primers targeted to the unique sequences flanking the microsatellite motif. PCR can easily be semi-automated (1 )
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) (1 ). PCR products are visualized by:
Fig. Tri-nucleotide repeat microsatellite marker: Expansion of the CGG triplet in the FMR-1 gene seen in the fragile X syndrome. Normal individuals have from 6 to 54 copies of the CGG
repeat, 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 200 to 1300 in individuals showing full symptoms of the disease.
PCR for semi-automated Microsatellite genotyping by auto-radiography.
Denaturated Poly-acrylamide gel electrophoresis (PAGE) for microsatellite genotyping
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 one end-labeled primer (labels one strand). Primers are endlabeled 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 black spots. A photon of light or a b particle or g ray released from a radioactive molecule "activate" silver bromide crystals on the film emulsion. This renders them capable of being reduced through the developing process to form silver metal (a "grain"). The silver grains on the film form the image. Co-amplification and/or co-loading of multiples microsatellites allows for multiplex genotyping (2-4 systems).
Agarose gel Electrophoresis (A), (B), (C).
Staining by ethidium Bromide (B), Stained by auto-radiograph (C).
Labeling of genetic markers
Labelling of dNTP by P32 radio-isotops in microsatellite tying by auto-radiogragy.
Production of PCR product with labeled P32
Mode of action of P32 labeling in PCR product.
Fluorescence labelling: The PCR products are labeled either by using primers or dNTPs which are tagged with an appropriate fluorophore, a chemical group which 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, and amino methyl coumarin, a blue fluorescent dye. Fluorophoes are characterized by their excitation and emission spectra. The PCR products are detected during migration using automatic sequencers. (1 ) Co-amplification and/or co-loading of multiples microsatellites allows for multiplex genotyping (up to 20 systems). (1 ) Software packages allow for semi-automated data capture (1 ). Microsatellite profiles are often difficult to read due to artefactual bands, which result from (1 ): the differential migration of the two DNA strands in denaturing acrylamide gels the "+ 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:
New era of molecular genetics: Using fluorescent labeled primers.(4Pictures of ABI377 PerkinElmer DNA sequencers)
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 nucleotide substitutions (transitions > transversions) or single nucleotide insertions/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:
1) Single Stranded Conformation Polymorphism (SSCP)(1 ) 2) Allele specific oligonucleotides (ASO)(1 ) 3) Single nucleotide polymorphic discrimination by an electronic dot blot assay (ASO) on semconductor microchips (1 ; 1 ) 4) Reverse dot blot on DNA chips (1 ) 5) Dynamic allele specific hybridisation (DASH) (1 ; 1 ) 6) Allele-specific PCR (=amplification refractory mutation system or ARMS test)(1 ) 7) Mutation detection the ARMS test in combination with the TaqmanTM 5' exonuclease assay (exploiting the 5'->3' exonuclease activity of Taq DNA polymerase).(1 ) 8) Minisequencing and analysis of the extension products by PAGE. 9) Minisequencing and analysis of the extension products on DNA chips 10) Minisequencing and analysis of the extension products using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF)(1 ) 11) Pyrosequencing (1 ) 12) OLA (1 ) 13) Invasive clivage of oligonucleotide probes (Invader technology)
1) Single Stranded Conformation Polymorphism (SSCP)(1 (Figure 1-3)
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 a GAG (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 specific for the mutant (b S) allele, being identical to the equivalent sequence of the mutant allele. The labeled ASOs can be individually hybridized to denatured genomic DNA samples on dot-blots. The b A- and b S-specific ASOs can hybridize 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-specific ASO and the b A allele have a single mismatch and are unstable at high hybridization stringency.
2) Single nucleotide polymorphic discrimination by an electronic dot blot assay (ASO) on semiconductor microchips
Single nucleotide polymorphic discrimination by an electronic dot blot assay (ASO) on semiconductor microchips
Correct base-pairing at the 3' end of PCR primers is the basis of allele-specific PCR.
The allele-specific oligonucleotide primers ASP1 and ASP2 are designed to be identical to the sequence of the two alleles over a region preceding the position of the variant nucleotide, up to and terminating in the variant nucleotide itself. ASP1 will bind perfectly to the 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 1 sequence, making amplification impossible. Similarly ASP2 can bind perfectly to allele 2 and initiate amplification, unlike ASP1.
Pyrosequencing
is sequencing by synthesis. ' A simple to use technique for accurate and consistent analysis of large numbers of short to medium length DNA sequences. Step1. A sequencing primer is hybridized to a single stranded, PCR amplified, DNA template, and incubated with the enzymes, DNA polymerase, ATP sulfurylase, luciferase and apyrase, and the substrates, adenosine 5 phosphosulfate (APS) and luciferin.
