Corso di Struttura e Funzione dei Genomi (LM7) mutuato con il corso di Genomica e Trascrittomica (LM9) Genomi. Diapositive, articoli e dispense:

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1 Corso di Struttura e Funzione dei Genomi (LM7) mutuato con il corso di Genomica e Trascrittomica (LM9) Genomi Diapositive, articoli e dispense:

2 Vettori di clonaggio I vettori di clonaggio sono brevemente trattati ai soli fini di un «ripasso» delle loro caratteristiche, dato che ad essi si fa riferimento in diversi argormenti trattati nell ambito del Corso Per lo studente con scarse conoscenze, informazioni dettagliate sono disponibili su: Tecnologie Biomolecolari 2010.pdf

3 Plasmidi

4 Cosmidi

5 Fagi

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7 Fagi

8 Yeast Artificial Chromosome (YAC)

9 BAC Il vettore BAC è costituito da: geni oris e repe (Mediano le replicazione unidirezionale del fattore F) geni para e parb (Mantengono il numero di copie del fattore ad un basso livello (1 copia).un alto numero di copie potrebbe causare problemi di instabilità degli inserti per delezioni e riarrangiamenti del DNA clonato a causa di eventi di ricombinazione omologa) gene per la resistenza ad un antibiotico sito di clonaggio: È fiancheggiato dai promotori T7 e SP6 che generano sonde di DNA per il chromosome walking e per il sequenziamento d'inserti di DNA. cosn e loxp sono siti di taglio specifici per terminasi del batteriofago l e consentono la formazione di estremità che possono essere usate, nelle mappe di restrizione, per riarrangiare i cloni. I cloni ricombinanti sono selezionati mediante colorazione bianco/blu.

10 Comparazione fra YAC and BAC Cloning System Features YAC BAC Configuration Linear Circular Host Yeast Bacteria Copy Number / Cell Cloning Capacity Unlimited up-to 350 kb Transformation Spheroplast (10^7 T/ug) Electroporation (10^10 T/ug) Chimerism up to 40% None to low DNA Isolation Pulsed-field-gelelectrophoresis - Gel Isolation Insert Stability Unstable Stable Standard Plasmid Miniprep

11 Mappe genetiche e fisiche Le mappe sono brevemente trattate ai soli fini di un «ripasso» delle loro caratteristiche, dato che ad esse si fa riferimento in diversi argormenti trattati nell ambito del Corso

12 Mappe fisiche e mappe genetiche Mappa genetica o Mappa di concatenazione: Rappresentazione della distanza che separa i geni, basata sui dati di ricombinazione genetica Mappa fisica: Rappresentazione della distanza che si basa sulla distanza fisica lungo il cromosoma (sovente espressa in bp) Relazione tra mappa genetica e mappa fisica La distanza della mappa genetica per le 3000Mb del genoma umano e' di circa 3000cM e pertanto 1cM corrisponde approssimativamente a una distanza di mappa fisica di 1Mb. In realta' il rapporto delle distanze di mappa genetica e fisica sui segmenti cromosomici spesso deviano da questo valore medio a causa della localizzazione non casuale dei chiasmi. I segmenti cromosomici contenenti gli "hot spots" di ricombinazione mostrano una piu' alta frequenza di ricombinazione e per tanto marcatori molecolari localizzati in questa zona sembrano piu' distanti del reale. In generale c'e' una frequenza di crossingover piu' alta in corrispondenza delle regioni subtelomeriche rispetto a quelle centromeriche.

13 Mappa genetica

14 Mappa fisica

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16 Overview genetic markers Molecular markers abundance level of polymorphism locus specificity codominance of alleles reproducibility Allozymes low low yes yes high low RFLP high medium yes yes high high Minisatellites medium high no/yes no/yes high high PCR-sequencing low low yes yes high high RAPD high medium no no low low Microsatellites high high yes yes high low ISSR medium-high medium no no medium-high low labor-intensity SSCP low low yes yes medium low-medium CAPS low low-medium yes yes high low-medium SCAR low medium yes yes/no high low AFLP high medium no no/yes high medium TaqMan low yes no/yes high low RAD

17 Isoenzymes ( Allozymes are allelic variants of enzymes encoded by structural genes. Enzymes are proteins consisting of amino acids, some of which are electrically charged. As a result, enzymes have a net electric charge, based on the stretch of amino acids comprising the protein. When due to mutation an amino acid has been replaced, the net electric charge of the protein may have been altered. Because changes in electric charge affect the migration rate of proteins in an electric field, allelic variation can be detected by gel-electrophoresis and subsequent specific enzymatic staining. Per enzyme usually two or more loci can be distinguished that have been termed isoloci. Therefore, allozyme variation is also referred to as isozyme variation. Synonyms Isozymes Analytical procedures Preparation of tissue homogenates Separation of the polymorphisms by polyacrylamide or starch gel-electrophoresis Visualization of the polymorphisms by specific enzymatic staining Main Requirements Laboratory setup for gel-electrophoresis Facilities/equipment to maintain gels at 2-5 C during electrophoresis Advantages No DNA extraction involved No primers or probes required Quick and easy to assay Low costs involved Zymograms can be interpreted in terms of loci and alleles Codominance of alleles High reproducibility Disadvantages Low abundance Low level of polymorphism Zymograms sometimes difficult to interpret due to complex banding profiles Proteins with identical electrophoretic mobility (co-migration) may not be homologous Profilo enzimatico influenzato dall ambiente (es. perossidasi indotte in condizioni di stress ossidativo)

18 RFLP (Restriction Fragment Length Polymorphism) RFLPs are fragments of restricted DNA (usually within the 2-10 kb range) separated by gel electrophoresis and detected by subsequent Southern blot hybridization to a radiolabeled DNA probe consisting of a sequence homologous to a specific chromosomal region. The locus specific probes, consisting of a sequence of unknown identity or part of the sequence of a cloned gene, are obtained by molecular cloning and isolation of suitable DNA fragments. Sequence variation affecting the occurrence (absence or presence) of endonuclease recognition sites is considered to be main cause of length polymorphisms. RFLPs are generally found to be moderately polymorphic and can be applied in comparisons ranging from the individual level to closely related species. Because of their high genomic abundance and random distribution throughout the genome, RFLPs have frequently been used in gene mapping studies. Analytical procedures Extraction of DNA Digestion of DNA by endonuclease restriction Separation of the fragments by agarose gel-electrophoresis Transference of the fragments to a nylon filter by Southern blotting Hybridization of fragments to a locus specific radiolabeled DNA probe Visualization of the polymorphisms by autoradiography Advantages High genomic abundance Random distribution throughout the genome Band profiles can be interpreted in terms of loci and alleles Codominance of alleles High reproducibility Filters can be washed and reprobed several times Disadvantages Large quantities of purified, high molecular weight DNA required (5-10 µg) Laborious and technically demanding High costs involved Additional development costs in case suitable probes are unavailable Not amenable to automation

19 RFLP (restriction fragment length polymorphism )

20 RFLP (restriction fragment length polymorphism )

21 CAPS (Cleaved Amplified Polymorphic Sequence) CAPS are DNA fragments amplified by the Polymerase Chain Reaction (PCR) using specific bp primers, followed by digestion with a restriction endonuclease. Subsequently, length polymorphisms resulting from variation in the occurrence of restriction sites are identified by gel-electrophoresis of the digested products. In comparison with RFLP-analysis, polymorphisms are more difficult to identify because of the limited size of the amplified fragments ( bp). CAPS-analysis, however, does not require time-consuming Southern blot hybridization and radioactive detection. CAPS have been applied predominantly in gene mapping studies. Analytical procedures Extraction of DNA Amplification of DNA fragments by PCR Digestion of the amplified fragments by endonuclease restriction Separation of the fragments by agarose or polyacrylamide gel-electrophoresis Visualization of the polymorphisms by ethidium-bromide staining and ultraviolet light (agarose gels) or silver staining (polyacrylamide gels) Advantages Low quantities of template DNA required ( ng per reaction) Band profiles can be interpreted in terms of loci and alleles Codominance of alleles High reproducibility Disadvantages Sequence data required for primer construction

22 marker B4 is a cleaved amplified polymorphism sequence (CAPS) marker.

23 RAPD (Random Amplified Polymorphic DNA) RAPDs are DNA fragments amplified by the Polymerase Chain Reaction (PCR) using short (generally 10 bp) synthetic primers of random sequence. These oligonucleotides serve as both forward and reverse primer and usually are able to amplify fragments from 3-10 genomic sites simultaneously. Amplified fragments (within the kb range) are separated by gel-electrophoresis and polymorphisms are detected as the presence or absence of bands of particular size. These polymorphisms are considered to be primarily due to variation in the primer annealing sites. RAPDs have been used for many purposes, ranging from studies at the individual level (e.g. genetic identity) to studies involving closely related species. Due to their very high genomic abundance, RAPDs have also been applied in gene mapping studies. Analytical procedures Extraction of DNA Amplification of DNA fragments by PCR Separation of the polymorphisms by agarose gel-electrophoresis (AP-PCR and DAF fragments are usually separated on polyacrylamide gels) Visualization of the polymorphisms by ethidium-bromide staining and ultraviolet light (AP-PCR and DAF fragments are usually visualized by silver staining or autoradiography) Advantages Low quantities of template DNA required (5-50 ng per reaction) No sequence data for primer construction required Low costs involved Very high genomic abundance Random distribution throughout the genome Generation of multiple bands per reaction Amenable to automation Disadvantages Purified, high molecular weight DNA required Precautions are needed to avoid contamination of DNA because short, random primers are used that are able to amplify DNA fragments in a variety of organisms Highly standardized experimental procedures are needed because of sensitivity to reaction conditions Dominance of alleles Low reproducibility Similar sized fragments may not be homologous

24 RAPD (Random amplified polymorphic DNA )

25 RAPD (Random amplified polymorphic DNA )

26 SCAR (Sequence Characterized Amplified Region) SCARs are DNA fragments amplified by the Polymerase Chain Reaction (PCR) using specific bp primers, designed from nucleotide sequences established in cloned RAPD (Random Amplified Polymorphic DNA) fragments linked to a trait of interest. By using longer PCR primers, SCARs do not face the problem of low reproducibility generally encountered with RAPDs. SCARs are locus specific and have been widely applied in gene mapping studies and marker assisted selection. Analytical procedures Extraction of DNA Amplification of DNA fragments by PCR Separation of the polymorphisms by agarose gel-electrophoresis Visualization of the polymorphisms by ethidium-bromide staining and ultraviolet light Advantages Low quantities of template DNA required ( ng per reaction) Easy and quick to assay High reproducibility Amenable to automation Disadvantages Sequence data required for primer construction

27 AFLP (Amplified Fragment Length Polymorphism) AFLP TM is a trademark of Keygene (Wageningen). AFLPs are DNA fragments ( bp) obtained from endonuclease restriction, followed by ligation of oligonucleotide adapters to the fragments and selective amplification by the Polymerase Chain Reaction (PCR). The PCR-primers consist of a core sequence (part of the adapter), a restriction enzyme specific sequence and 1-3 selective nucleotides. The AFLP-technique simultaneously generates fragments from many genomic sites (usually fragments per reaction) that are separated by gel-electrophoresis and generally scored as a dominant marker. However, by using automatic gel scanners, heterozygotes may be distinguished from homozygotes based on band intensity differences. Because of the highly informative fingerprinting profiles generally obtained, AFLPs can be applied in studies involving genetic identity, parentage and identification of clones and cultivars. Due to their high genomic abundance and random distribution throughout the genome, AFLPs are also considered relevant markers in gene mapping studies. Analytical procedures Extraction of DNA Digestion of DNA by endonuclease restriction and ligation of oligonucleotide adapters to the DNA fragments Selective amplification of part of the DNA fragments Separation of the fragments by polyacrylamide gel-electrophoresis Visualization of the polymorphisms by autoradiography, silver staining or fluorescence Advantages No sequence data for primer construction required High genomic abundance Random distribution throughout the genome, although clustering around centromers has been reported Generation of many informative bands per reaction High reproducibility Amenable to automation Disadvantages Purified, high molecular weight DNA required Dominance of alleles Similar sized fragments may not be homologous

28 Amplified Fragment Length Polymorphisms (AFLPs)

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30 SSCP (Single-Strand Conformation Polymorphism) SSCPs are DNA fragments ( bp) amplified by the Polymerase Chain Reaction (PCR) using specific bp primers, followed by gel-electrophoresis of single-strand DNA to detect nucleotide sequence variation. The method is based on the fact that the electrophoretic mobility of single-strand DNA highly depends on the secondary structure (conformation) of the molecule, which changes significantly in case of mutation. SSCP provides a method to detect nucleotide variation without the need to sequence DNA samples. SSCPs have been applied to detect mutations in genes using gene sequence information for primer construction. Related techniques DGGE (Denaturing Gradient Gel Electrophoresis): separation of DNA fragments due to mobility differences under increasing denaturing conditions (formamide/urea concentrations) TGGE (Thermal Gradient Gel Electrophoresis): similar to DGGE, but subjected to heath-denaturing conditions Analytical procedures Extraction of DNA Amplification of DNA fragments by PCR Denaturation of the amplified fragments to a single-strand form Separation of the polymorphisms by polyacrylamide gel-electrophoresis Visualization of the polymorphisms by silver staining or autoradiography Advantages Low quantities of template DNA required ( ng per reaction) Band profiles can be interpreted in terms of loci and alleles Codominance of alleles Disadvantages Sequence data required for primer construction Highly standardized electrophoretical conditions are needed to obtain reproducible results Absence of mutation cannot be proven because some mutations may remain undetected

31 Denaturing Gradient Gel Electrophoresis (DGGE) PCR primer with a 5' tail consisting of a sequence of 40 GC!

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33 Microsatellites Microsatellites are molecular marker loci consisting of tandem repeat units of very short (1-5 basepairs) nucleotide motif. In case the nucleotide sequences in the flanking regions of the microsatellite are known, specific primers (generally bp) can be designed to amplify the microsatellite by the Polymerase Chain Reaction (PCR). Polymerase slippage during DNA replication (or slipped strand mispairing) is considered to be the main cause of variation in the number of repeat units, resulting in length polymorphisms that can be detected by gel-electrophoresis. Due to their high level of polymorphism, microsatellites are informative markers that can be used for many population genetic purposes, ranging from the individual level (e.g. clone and strain identification) to closely related species. Synonyms SSLP (Simple Sequence Length Polymorphisms); SSR (Simple Sequence Repeats); STMS (Sequence Tagged Microsatellites) Analytical procedures Extraction of DNA Amplification of DNA fragments by PCR Separation of the polymorphisms by polyacrylamide Visualization of the polymorphisms by autoradiography, silver staining or fluorescence (polyacrylamide gels), or ethidium-bromide staining and ultraviolet light (agarose gels) Advantages Low quantities of template DNA required ( ng per reaction) High genomic abundance Random distribution throughout the genome High level of polymorphism Codominance of alleles Allele sizes can be determined with an accuracy of 1 bp, allowing accurate comparison across different gels High reproducibility Different microsatellites may be multiplexed in PCR or on gel Wide range of applications Amenable to automation Disadvantages High development costs in case primers are not yet available Heterozygotes may be misclassified as homozygotes when null-alleles occur due to mutation in the primer annealing sites

34 Microsatelliti Microsatellite DNA is thus a class of repetitive DNA based on dinucleotide repeats. The most common type consists of repeats of CA and its complement GT, as in the following example:

35 Figura SSR When the repeating unit is less than four, the VNTR is called a microsatellite and when the repeating unit is longer it is a minisatellite. I microsatelliti sono anche chiamati Simple Sequence Repeat (SSR)

36 Minisatellites Disadvantages Large quantities of purified, high molecular weight DNA required (5-10 µg) Laborious and technically demanding High costs involved Distribution across the genome may be non-random Similar sized fragments may not be homologous (in case of multilocus probes) Difficulties in comparing polymorphisms across different gels Minisatellites are molecular marker loci consisting of tandem repeat units of a base motif, flanked by conserved endonuclease restriction sites. They are detected by gel electrophoresis of restricted DNA and subsequent Southern blot hybridization to a radiolabeled DNA probe containing multiple copies of the minisatellite core sequence. A minisatellite profile consisting of many bands (within the 4-20 kb range) is generated by using common multilocus probes that are able to hybridize to minisatellite sequences in different species. Locus specific probes can be developed by molecular cloning of DNA restriction fragments and subsequent screening with a multilocus minisatellite probe. Variation in the number of repeat units is considered to be the main cause of length polymorphisms. Due to the high mutation rate of minisatellites, the level of polymorphism is substantial, generally resulting in unique multilocus profiles. Therefore, minisatellites are particularly useful in studies involving genetic identity, parentage, clonal growth and structure, and identification of varieties and cultivars Synonyms DNA fingerprinting; VNTR (Variable Number of Tandem Repeats) Analytical procedures Extraction of DNA Digestion of DNA by endonuclease restriction Separation of the fragments by agarose gel-electrophoresis Transference of the fragments to a nylon filter by Southern blotting Hybridization of fragments to a radiolabeled minisatellite probe Visualization of the polymorphisms by autoradiography Advantages High level of polymorphism Generation of many informative bands per reaction (in case of multilocus probes) High reproducibility

37 Obtaining a DNA fingerprint by using a VNTR probe. (a) Preparation of the probe. The first intron of the myoglobin gene has four repeats of the sequence shown, which contains a 13- bp core sequence (shown in boldface). This core sequence is found at other VNTR loci, labeled VNTR I, II, and III in this simple diagrammatic representation. (b) The number of repeats at the three VNTR loci with the core sequence. The Southern blot has been probed with the 33-bp repeat in part a and shows the DNA fingerprints of three people.

38 Fig. 5 Détection des réarrangements du minisatellite CEB1-1.8 et CEB1-0.6 dans les cellules rad27δ de S. cerevisiae. L'instabilité se manifeste par l'apparition de variants de nouvelles tailles, plus courtes ou plus longues que celles des allèles parentaux (indiqués par des flèches).

39 SNP

40 SNP (single nucleotide polymorphism Is a DNA sequence variation occurring when a single nucleotide A, T, C, or G in the genome (or other shared sequence) differs between members of a species (or between paired chromosomes in an individual). For example, two sequenced DNA fragments from different individuals, AAGCCTA to AAGCTTA, contain a difference in a single nucleotide. In this case we say that there are two alleles : C and T. Almost all common SNPs have only two alleles. Variations in the DNA sequences of humans can affect how humans develop diseases and respond to pathogens, chemicals, drugs, vaccines, and other agents. SNPs are also thought to be key enablers in realizing the concept of personalized medicine. [3] However, their greatest importance in biomedical research is for comparing regions of the genome between cohorts (such as with matched cohorts with and without a disease). The study of single-nucleotide polymorphisms is also important in crop and livestock breeding programs

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42 Advantages and disadvantages of various DNA fingerprinting methods Type Advantages Disadvantages Isozymes RAPD AFLP SSR RFLP SNP Inexpensive Co-dominant Expressed genes Inexpensive Simple High discrimination Many markers High discrimination Highly reproducible Co-dominant Characterized DNA Highly reproducible Co-dominant Characterized DNA High discrimination Co-dominant The small number of isozymes available can lead to poor discrimination capacity between samples. Poor resolution of electrophoretic bands. Markers can be difficult to differentiate because of complex banding patterns of oligomeric isozymes and co-migration in electrophoresis Uncharacterized DNA which is amplified in RAPD provides no additional information, which is sometimes a useful property of other marker systems. Low stringency of the PCR process used in RAPD can result in profile reproducibility problems. Unknown gene function Expensive Complex procedure Unknown gene function Complex procedure to establish initially for given taxa. Expensive (unless using extant SSR primers). Taxaspecific, there are limitations to the transferability of SSR markers between taxa; only successful with closely related genera Unknown gene function (unless EST-derived) Complex procedure Laborious Complex to establish initially for given taxa

43 Restriction site Associated DNA (RAD) markers Restriction site associated DNA (RAD) markers can be identified and typed by detecting differential hybridization patterns of RAD tags on a microarray. Genomic DNA samples S1 and S2 contain the recognition sequence for various restriction enzymes at locations throughout the genome. Dark blue triangles represent restriction sites of a particular enzyme. Some of these restriction sites are only present in one sample because of polymorphisms that disrupt the recognition sequence (red asterisks). The two samples are separately digested with a particular restriction enzyme and then ligated to biotinylated linkers (light blue ellipses). The DNA is randomly sheared leaving only the fragments that were directly flanking a restriction site attached to biotin linkers. These fragments are purified using streptavidin beads and released by digestion at the original restriction site. Loci containing polymorphisms, such as the left locus of S2 or the right locus of S1, will not contain tags for that locus in the purified RAD-tag sample, thus resulting in differential hybridization patterns of RAD tags on a microarray. Miller M R et al. Genome Res. 2007;17:

44 Enriched RAD marker microarray production and characterization Enriched RAD marker microarray production and characterization. RAD-tag samples S1 and S2 contain polymorphic sets of RAD tags. RAD tags that are present in both individuals will not serve as informative markers. In order to produce an array that types a large number of informative markers, subtractive hybridization is used to enrich for sample-specific RAD tags. RAD-tag clone libraries are generated from these enriched samples. These clone libraries are used as templates for PCR, the products of which are spotted to produce RAD marker microarrays. To identify informative markers, RAD-tag samples S1 and S2 are fluorescently labeled and competitively hybridized directly against each other to the array. Miller M R et al. Genome Res. 2007;17:

45 The process of RADSeq The process of RADSeq (A) Genomic DNA is sheared with a restriction enzyme of choice (SbfI in this example). (B) P1 adapter is ligated to SbfI-cut fragments. The P1 adapter is adapted from the Illumina sequencing adapter (full sequence not shown here), with a molecular identifier (MID; CGATA in this example) and a cut site overhang at the end (TGCA in this example). (C) Samples from multiple individuals are pooled together and all fragments are randomly sheared. Only a subset of the resulting fragments contains restriction sites and P1 adapters. (D) P2 adapter is ligated to all fragments. The P2 adapter has a divergent end. (E) PCR amplification with P1 and P2 primers. The P2 adapter will be completed only in the fragments ligated with P1 adapter, and so only these fragments will be fully amplified. (F) Pooled samples with different MIDs are separated bioinformatically and SNPs called (C/G SNP underlined). (G) As fragments are sheared randomly, paired end sequences from each sequenced fragment will cover a bp region downstream of the restriction site. Davey J W, Blaxter M L Briefings in Functional Genomics 2010;9:

46 Gene tagging Transposon tagging T-DNA tagging Activation tagging

47 Activation TAGGING Transposon tagging

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50 TAIL-PCR is very simple, efficient, and highly specific. Since no other manipulations apart from PCR are required, TAIL-PCR is especially suitable for isolation of targeted unknown sequences from a large number of samples. This technique has been used to recover insert ends from rice BAC clones for chromosome walking and mapping.

51 AIMS (Amplification of insertion mutagenised sites)

52 Amplification of insertion mutagenised sites (AIMS)

53 Reverse genetics Reverse genetics is an approach to discovering the function of a gene that proceeds oppositely to how such discoveries typically unfold in classical genetics, or in forward genetics. By the classical approach, geneticists first look for rare individuals with unusual traits or phenotypes, and then they trace these traits to an underlying faulty allele or gene. Locating the gene on its chromosome is the end point of an investigation. With the readily performed modern techniques of DNA sequencing and as a result of the sequencing of many whole genomes, many genetic sequences are discovered in advance of any other information about them. To learn the influence a sequence has on phenotype, or to discover its biological function, researchers may engineer a: Change or disruption by site-directed mutagenesis, for example, or by deletion of a gene by gene knockout (as can be done in some organisms, such as yeast and mice) -and only afterwards look for the effect of such alterations in the whole organism. The discovery of gene silencing using double stranded RNA also known as RNA interference has also made this approach very promising. A third reverse genetics technique is the creation of transgenic organisms that overexpress a gene of interest. The resulting phenotype may reflect the normal function of the gene.