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Marcadores Moleculares

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    1. Marcadores Moleculares Introduo e Histrico Descrio de Marcadores Comparao entre Marcadores Moleculares Classificao de Marcadores Moleculares Caractersticas do Genoma de Planta Aplicaes diversidade gentica mapeamento e seleo assistida

    2. INTRODUO E HISTRICO Marcadores Moleculares

    3. Gentica arte e seleo inconsciente da inveno da agricultura at sc. XIX 1900s - Descoberta dos princpios genticos 1920-50 - Melhoramento gentico cientfico gentica quantitativa e biometria (fentipo previsor ruim do valor gentico!) 1970-80 - Utilizao de marcadores genticos moleculares

    4. Sucesso no melhoramento depende da capacidade de distinguir fatores genticos herdveis dos ambientais Marcadores genticos so unidades herdveis simples

    5. Polimorfismo de DNA resulta acmulo de mutaes pontual ou insero/deleo macro-rearranjos: translocaes, inverses, delees

    7. Histrico de Marcadores 1. Karl Sax (1923): props mtodo para localizao de QTLs ligao entre genes de caracterstica qualitativa (cor de semente) e quantitativa (peso de semente); Problema: ausncia de mutaes mltiplas em estoque de elite, baixa viabilidade 2. Hunter & Markert (1957) - marcas bioqumicas desenvolveram isoenzimas em gel de amido

    8. Histrico de Marcadores 3. Hubby & Lewotin (1966) demonstraram que 30% de loci de isoenzimas exibiam polimorfismo em populaes selvagens de Drosophila; 4. 1970s - ferramentas moleculares desenvolvimento de vetores de clonagem; enzimas de restrio; polimerases; ligases; Southern (1977);

    9. Histrico de Marcadores 5. RFLP proposto por Botstein et al. (1980) descrito para humanos 6. PCR proposto por Mullis & Faloona (1987) 7. VNTR por Jeffrey (1987) 8. RAPD por Rafalski et al. (1990)

    10. Histrico de Marcadores 9. SSR em plantas por Akkaya et al. (1992) 10. AFLP por Zabeau & Vos (1993) 11. CAPS por Konieczny & Ausubel (1993) 12. SCAR por Paran & Michelmore (1993) 13. Cho et al. (1999) - SNPs em Arabidopsis

    11. DESCRIO DOS MARCADORES MOLECULARES

    12. Marcadores Moleculares RFLP VNTR (minissatlite) RAPD, AP-PCR, DAF PCR-especfico - SSR, ISSR, CAPS, SCARs AFLP SNPs

    13. Restriction Fragment Length Polymorphism - RFLP RFLP examina diferena em tamanho de fragmentos de restrio de DNA especficos Polimorfismo deriva de mutao pontual, insero, deleo Utiliza-se DNA celular total Requer DNA puro de alto peso molecular

    14. Metodologia de RFLP 1 . Digerir DNA em fragmentos pequenos 2. Separao dos fragmentos por gel eletroforese 3. Transferncia de fragmentos de DNA para filtro

    15. Metodologia de RFLP 4. Visualizao dos fragmentos de DNA sondas marcadas (32P) ou a frio 5. Anlise dos resultados bandas analisadas para alelos e/ou presena/ausncia diferenas em padro de bandas reflete diferenas genticas A escolha de sonda/enzima de restrio crucial

    16. Digesto de DNA Genmico e Separao em Gel

    17. Transferncia para Membrana de Nylon ou Nitrocelulose

    18. Hibridizao em Nylon ou Nitrocelulose

    19. Construo de biblioteca genmica ou de cDNA

    22. Interpretao de resultados

    26. Herana de RFLPs

    27. Anlise de Diversidade e Filogenia por RFLP

    28. RFLP: sondas de locos nico DNA Nuclear biblioteca genmica biblioteca de cDNA DNA Citoplasmtico biblioteca de DNA cloroplstico e mitocondrial Sondas de RFLP so: locos-especfica, co-dominante espcie-especfica

    29. RFLP: sondas multi-locus Repeties em linha (tandem) - til encontrada em vrios loci altamente polimrficas Sequncia de Minissatlite VNTR: variable number of tandem repeats uso em DNA fingerprinting uso de seqncias repetidas de fago M13

    30. Interpretao dos resultados

    31. Vantagens e Desvantagens de RFLP Reprodutvel Marcadores co-dominantes Simples Trabalhoso Caro Uso de sondas radioativas*

    32. Random Amplification of Polymorphic DNA - RAPD Amplifica seqncias annimas de DNA usando primers arbitrrios 10 bases com >50% G+C PCR com um nico primer Mtodo rpido para deteco de polimorfismos Marcador dominante Problemas de reproducibilidade The random amplified polymorphic DNA (RAPD) technique is a PCR based method which uses one or sometimes two short arbitrary primers (usually 8-10 bases) to amplify anonymous stretches of DNA which are then separated and visualised by gel electrophoresis. The key point about this technique is that nothing is known about the identity of the amplification products. The amplification products are however extremely useful as markers in genetic diversity studies. Other important features of the technique are: The number of fragments. Many different fragments are normally amplified using each single primer, and the technique has therefore proved a fast method for detecting polymorphisms. The majority of commercially produced primers result in 6 to 12 fragments; some primers may fail to give any amplification fragments from some material. Simplicity of the technique. RAPD analysis does not involve hybridisation/autoradiography or high technical expertise. Only tiny quantities of target DNA are required. Arbitrary primers can be purchased. Unit costs per assay are low. This has made RAPD analysis very popular. RAPD markers are dominant. Amplification either occurs at a locus or it does not, leading to scores of band presence/absence; this means that homozygotes and heterozygotes cannot be distinguished. Problems of reproducibility - RAPD does suffer from a sensitivity to changes in PCR conditions resulting in changes to some of the amplified fragments. Reproducible results can be obtained if care is taken to standardise the conditions used (Munthali et al., 1992; Lowe et al., 1996). The random amplified polymorphic DNA (RAPD) technique is a PCR based method which uses one or sometimes two short arbitrary primers (usually 8-10 bases) to amplify anonymous stretches of DNA which are then separated and visualised by gel electrophoresis. The key point about this technique is that nothing is known about the identity of the amplification products. The amplification products are however extremely useful as markers in genetic diversity studies. Other important features of the technique are: The number of fragments. Many different fragments are normally amplified using each single primer, and the technique has therefore proved a fast method for detecting polymorphisms. The majority of commercially produced primers result in 6 to 12 fragments; some primers may fail to give any amplification fragments from some material. Simplicity of the technique. RAPD analysis does not involve hybridisation/autoradiography or high technical expertise. Only tiny quantities of target DNA are required. Arbitrary primers can be purchased. Unit costs per assay are low. This has made RAPD analysis very popular. RAPD markers are dominant. Amplification either occurs at a locus or it does not, leading to scores of band presence/absence; this means that homozygotes and heterozygotes cannot be distinguished. Problems of reproducibility - RAPD does suffer from a sensitivity to changes in PCR conditions resulting in changes to some of the amplified fragments. Reproducible results can be obtained if care is taken to standardise the conditions used (Munthali et al., 1992; Lowe et al., 1996).

    33. RAPD The random amplified polymorphic DNA (RAPD) technique is a PCR based method which uses one or sometimes two short arbitrary primers (usually 8-10 bases) to amplify anonymous stretches of DNA which are then separated and visualised by gel electrophoresis. The key point about this technique is that nothing is known about the identity of the amplification products. The amplification products are however extremely useful as markers in genetic diversity studies. Other important features of the technique are: The number of fragments. Many different fragments are normally amplified using each single primer, and the technique has therefore proved a fast method for detecting polymorphisms. The majority of commercially produced primers result in 6 to 12 fragments; some primers may fail to give any amplification fragments from some material. Simplicity of the technique. RAPD analysis does not involve hybridisation/autoradiography or high technical expertise. Only tiny quantities of target DNA are required. Arbitrary primers can be purchased. Unit costs per assay are low. This has made RAPD analysis very popular. RAPD markers are dominant. Amplification either occurs at a locus or it does not, leading to scores of band presence/absence; this means that homozygotes and heterozygotes cannot be distinguished. Problems of reproducibility - RAPD does suffer from a sensitivity to changes in PCR conditions resulting in changes to some of the amplified fragments. Reproducible results can be obtained if care is taken to standardise the conditions used (Munthali et al., 1992; Lowe et al., 1996). The random amplified polymorphic DNA (RAPD) technique is a PCR based method which uses one or sometimes two short arbitrary primers (usually 8-10 bases) to amplify anonymous stretches of DNA which are then separated and visualised by gel electrophoresis. The key point about this technique is that nothing is known about the identity of the amplification products. The amplification products are however extremely useful as markers in genetic diversity studies. Other important features of the technique are: The number of fragments. Many different fragments are normally amplified using each single primer, and the technique has therefore proved a fast method for detecting polymorphisms. The majority of commercially produced primers result in 6 to 12 fragments; some primers may fail to give any amplification fragments from some material. Simplicity of the technique. RAPD analysis does not involve hybridisation/autoradiography or high technical expertise. Only tiny quantities of target DNA are required. Arbitrary primers can be purchased. Unit costs per assay are low. This has made RAPD analysis very popular. RAPD markers are dominant. Amplification either occurs at a locus or it does not, leading to scores of band presence/absence; this means that homozygotes and heterozygotes cannot be distinguished. Problems of reproducibility - RAPD does suffer from a sensitivity to changes in PCR conditions resulting in changes to some of the amplified fragments. Reproducible results can be obtained if care is taken to standardise the conditions used (Munthali et al., 1992; Lowe et al., 1996).

    34. Interpretao de RAPDs Marcadores RAPD so annimos Dados binrios (presena x ausncia) RAPD so dominantes (AA = Aa) Problemas de co-migrao mesma banda, mesmo fragmento? uma banda, um fragmento? Questionamento para filogenia banda homlogas?

    35. PCR com primers arbitrrios: acmulo de siglas! RAPD Random Amplified Polymorphic DNA DAF DNA Amplification Fingerprinting AP-PCR Arbitrarily Primed Polymerase Chain Reaction MAAP Multiple Arbitrary Amplicon Profiling (sugerido por incluir todas as pequenas variaes na tcnica) All of the following techniques use one or two, short, GC-rich primers of arbitrary sequence. RAPD was the first to become available (Williams et al., 1990) and is by far the most commonly used of these techniques. DAF - DNA amplication fingerprinting Differences between DAF (Caetano-Anolles, et al., 1991a,b) and RAPD: higher primer concentrations in DAF shorter primers used in DAF (5-8 nucleotides) two-temperature cycle in DAF compared to 3-temperature cycle in RAPD DAF usually produces very complex banding patterns AP-PCR - arbitrarily primed polymerase chain reaction Differences between AP-PCR (Welsh and McClelland, 1990) and RAPD: in AP-PCR the amplification is in three parts each with its own stringency and concentrations of constituents high primer concentrations are used in the first PCR cycles primers of variable length, and often designed for other purposes are arbitrarily chosen for use (e.g. M13 universal sequencing primer) MAAP is only an acronym proposed by Caetano-Anolles et al. (1992) to encompass all of these closely related techniques, but which is not commonly used.All of the following techniques use one or two, short, GC-rich primers of arbitrary sequence. RAPD was the first to become available (Williams et al., 1990) and is by far the most commonly used of these techniques. DAF - DNA amplication fingerprinting Differences between DAF (Caetano-Anolles, et al., 1991a,b) and RAPD: higher primer concentrations in DAF shorter primers used in DAF (5-8 nucleotides) two-temperature cycle in DAF compared to 3-temperature cycle in RAPD DAF usually produces very complex banding patterns AP-PCR - arbitrarily primed polymerase chain reaction Differences between AP-PCR (Welsh and McClelland, 1990) and RAPD: in AP-PCR the amplification is in three parts each with its own stringency and concentrations of constituents high primer concentrations are used in the first PCR cycles primers of variable length, and often designed for other purposes are arbitrarily chosen for use (e.g. M13 universal sequencing primer) MAAP is only an acronym proposed by Caetano-Anolles et al. (1992) to encompass all of these closely related techniques, but which is not commonly used.

    36. Diferenas entre ensaios com primers arbitrrios RAPD 10mers, gel de agarose corado com brometo DAF 5mers, gel de acrilamida e reao marcada 32P AP-PCR 10mers, gel de acrilamida e reao marcada 32P All of the following techniques use one or two, short, GC-rich primers of arbitrary sequence. RAPD was the first to become available (Williams et al., 1990) and is by far the most commonly used of these techniques. DAF - DNA amplication fingerprinting Differences between DAF (Caetano-Anolles, et al., 1991a,b) and RAPD: higher primer concentrations in DAF shorter primers used in DAF (5-8 nucleotides) two-temperature cycle in DAF compared to 3-temperature cycle in RAPD DAF usually produces very complex banding patterns AP-PCR - arbitrarily primed polymerase chain reaction Differences between AP-PCR (Welsh and McClelland, 1990) and RAPD: in AP-PCR the amplification is in three parts each with its own stringency and concentrations of constituents high primer concentrations are used in the first PCR cycles primers of variable length, and often designed for other purposes are arbitrarily chosen for use (e.g. M13 universal sequencing primer) MAAP is only an acronym proposed by Caetano-Anolles et al. (1992) to encompass all of these closely related techniques, but which is not commonly used.All of the following techniques use one or two, short, GC-rich primers of arbitrary sequence. RAPD was the first to become available (Williams et al., 1990) and is by far the most commonly used of these techniques. DAF - DNA amplication fingerprinting Differences between DAF (Caetano-Anolles, et al., 1991a,b) and RAPD: higher primer concentrations in DAF shorter primers used in DAF (5-8 nucleotides) two-temperature cycle in DAF compared to 3-temperature cycle in RAPD DAF usually produces very complex banding patterns AP-PCR - arbitrarily primed polymerase chain reaction Differences between AP-PCR (Welsh and McClelland, 1990) and RAPD: in AP-PCR the amplification is in three parts each with its own stringency and concentrations of constituents high primer concentrations are used in the first PCR cycles primers of variable length, and often designed for other purposes are arbitrarily chosen for use (e.g. M13 universal sequencing primer) MAAP is only an acronym proposed by Caetano-Anolles et al. (1992) to encompass all of these closely related techniques, but which is not commonly used.

    37. RAPD - resumo Rpido Simples Baixo custo Sem uso de radio-istopos Marcador dominante Problemas de reproducibilidade Problemas de interpretao

    40. Stio de Seqncia Dirigida (Sequence-tagged sites) Sequence-Tagged Microssatlites (STMS) ou SSR ou Microssatlites Microssatlites ancorados Inter-Simple Sequence Repeat (ISSR) Sequence-characterized amplified regions (SCARs) Cleaved amplified polymorphic sequence (CAPS) PCR-RFLP More and more sequence information is becoming available from different sources and can be located in widely available databases. This information is extremely useful for developing new strategies for the analysis of genetic variation. A sequence-tagged site (STS) is the general term given to a marker which is defined by its primer sequences (Olsen et al., 1989). STSs have been used extensively for mapping of the human genome. Examples of STSs are given in the following slides, namely: Sequence-tagged microsatellites (STMS) also known as Simple Sequence Repeat Polymorphisms (SSRP) Anchored microsatellite oligonucleotides including inter-simple sequence repeat (ISSR) primers Sequence-characterised amplified regions (SCARs) Cleaved amplified polymorphic sequence (CAPS)More and more sequence information is becoming available from different sources and can be located in widely available databases. This information is extremely useful for developing new strategies for the analysis of genetic variation. A sequence-tagged site (STS) is the general term given to a marker which is defined by its primer sequences (Olsen et al., 1989). STSs have been used extensively for mapping of the human genome. Examples of STSs are given in the following slides, namely: Sequence-tagged microsatellites (STMS) also known as Simple Sequence Repeat Polymorphisms (SSRP) Anchored microsatellite oligonucleotides including inter-simple sequence repeat (ISSR) primers Sequence-characterised amplified regions (SCARs) Cleaved amplified polymorphic sequence (CAPS)

    41. Microssatlites (SSR) Sequence-Tagged Microsatlites (STMS) tambm conhecido como microssatlite ou Simple Sequence Repeat (SSR) Normalmente locus simples e multi-allico Co-dominante Altamente reprodutvel

    42. Microssatlites STMS ou SSRs Seqncias curtas (1 a 6 bases) repetidas em tandem Presentes em procariotos e eucariotos Presentes em regies codificantes e no codificantes Maioria das repeties so dinucleotdeos (AC) n (AG) n (AT)n

    43. Polimorfismo devido a diferenas no nmero de repeties Escorregamento da DNA polimerase durante a replicao Crossing-over desigual entre cromtides irms Codominantes Normalmente locos simples e multi-allico Microssatlites

    44. Microssatlites (SSR) altamente informativo - vrios alelos por locos deteco por PCR facilmente transfervel entre labs distribuio homognea no genoma

    45. Microssatlites (SSR)

    46. Microssatlites (SSR) Obteno de seqncias: a partir de banco de dados de genoma ou cDNA hibridao com biblioteca genmica, identificao de clones e seqenciamento construo de biblioteca enriquecida por afinidade com seqncia da matriz

    47. Deteco do polimorfismo Gis de agarose Gis de acrilamida (detecta diferenas de at 2pb) colorao direta: nitrato de prata (barato) Colorao indireta: marcao radioativa ou fluorescente Microssatlites

    48. Problemas Custo e trabalho envolvidos no desenvolvimento dos primers Construo de bibliotecas genmica sequenciamento Triagem dos melhores primers ?Possibilidade de se usar seqncias depositadas em banco de dados EST SSR funcional x SSR genmico Microssatlites

    49. Microssatlites (SSR)

    55. Microssatlites (SSR)

    56. Microssatlites (SSR)

    58. Microssatlites Bananeira 3x e 4x Cir 24.25

    59. Microssatlites

    60. Microssatlites Ancorados ISSR Amplificao de segmentos genmicos flanqueados por repeties Anelamento locos-especfico Inter-simple sequence repeats (ISSR) ancorados na extremidade 3 ou 5 Marcadores dominantes Microssatlites mais teis que minissatlites

    61. Microssatlites Ancorados ISSR

    62. ISSR UBC 811 UBC 816

    63. SCARs SCARs - sequence-characterised amplified regions proposto por Paran & Michelmore (1993) marcador locus-nico derivado de fragmentos sequenciados de RAPD, ISSR, AFLP maior estabilidade - primers especficos analisado para presena/ausncia possibilidade de simplificao de anlise e automao

    64. SCARs

    66. CAPS ou PCR-RFLP CAPS - cleaved amplified polymorphic sequence marcador locus-especfico produto amplificado por PCR e analisado por RFLP seqncia de banco de dados, clones de cDNA ou genmico codominante

    67. CAPS

    70. Amplified Fragment Length Polymorphism - AFLP Combinao de RFLP e PCR Resulta em padres muito informativos Marcador dominante Mtodo cada vez mais usado

    74. AFLP de cana com 33P

    75. AFLP de feijo gel desnaturante corado com prata

    77. COMPARAO ENTRE MARCADORES MOLECULARES

    78. Escolha de Marcadores Caracterstica RFLP RAPD SSR AFLP ISSR CAPS Polimorfismo Pontual Pontual # Pontual Pontual Pontual InDel InDel Rep. InDel InDel InDel Nvel de Polimorfismo mdio mdio alto mdio mdio baixo Abundncia alta m.alta mdia m.alta mdia alta Dominncia CoDom Dom CoDom Dom Dom CoDom [DNA] 10 mg 25 ng 50 ng 500 ng 25 ng 25 ng Seqncia no no sim no no sim Marcao sim/no no no sim/no no no Repetibilidade alta baixa alta mdia baixa alta

    79. CLASSIFICAO DE MARCADORES MOLECULARES

    80. Classificao por Tipo de Tcnica Mtodos sem uso de PCR RFLP VNTR Mtodos com uso de PCR PCR com primers arbitrrios RAPD, AP-PCR, DAF, MAAP; Polimorfismo de Tamanho de Fragmento Amplificado AFLP; ISSR PCR stio-especfico CAPS, SCAR SSRs (microssatlites) TGGE, SSCP, DGGE

    81. Classificao por Nmero de Cpias da Seqncia Alvo Seqncia de poucas cpias - codificante RFLP Seqncia com cpias repetidas VNTR SSRs (microssatlites) ISSR Seqncia com nmero de cpias indefinido RAPD, AP-PCR, DAF, MAAP; AFLP; CAPS, SCAR