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. La-conformation-de-l, ADN est initialement en double brin, puis une première étape ?de chauffage (~95°C) permet de séparer les 2 brins d'ADN complémentaires

A. Vii, . D. Biais, and . Du-materiel-genetique-la-diversité-d, une communauté est mesurée par la richesse et la distribution des populations au sein de cette communauté. Ces deux paramètres sont estimés par le nombre de séquences génétiques spécifiques des populations découvertes dans l'échantillon et leur fréquence relative. Cependant, la validité de l'estimation de la diversité basée sur l'analyse des métagénomes dépend de la représentativité du matériel génétique extrait, 2000.

L. Qualité and . La-quantité-de-l-spiegelman, ADN métagénomique ou de l'ARN influence la structure microbienne observée (Theron et Cloete, 2000.

T. and C. Malik, Les bactéries ont des sensibilités différentes aux agents de lyse selon le type de parois qu'elles possèdent En effet, les bactéries Gram négatif, qui possèdent une couche de peptidoglycane plus fine que les bactéries Gram positif, sont plus sensibles à la lyse. Une lyse bactérienne insuffisante entraîne donc un taux de récupération de l'ADN ou de l'ARN des bactéries Gram négatif supérieur à celui des bactéries Gram positif. L'ADN ou l'ARN extrait est donc représentatif des bactéries à Gram négatif mais sous-estime la quantité de bactéries Gram + dans l'échantillon, Les méthodes de lyse bactérienne sont pour la plupart une combinaison de dégradation par voie chimique et physique La mesure de la biodiversité est ainsi biaisée avec une sous-estimation des bactéries Gram + et une aberration dans les proportions observées, 2000.

L. Quantité and . La-qualité-de-l, ADN ou ARN extrait sont aussi influencées par le type d'échantillon étudié. Pendant la lyse bactérienne, le matériel génétique libéré peut être adsorbé sur des particules présentes dans l'échantillon et ne pas être extrait par la suite, 2000.

L. Lyse, extraction de l'ADN sont aussi déterminantes pour le bon déroulement de l'amplification du matériel génétique par PCR

P. La, Polymerase Chain Reaction) est une technique d'amplification de l'ADN. Cette technique est basée sur l'utilisation d'une enzyme, la Taq polymérase qui copie la séquence d'ADN. L'amplification de l'ADN par la polymérase n'est possible que si des amorces (courtes séquences oligonucléotidiques de synthèse) se fixent sur l'ADN et permettent l

. Acinas, certains biais de la PCR sont intrinsèques à la nature même de l'enzyme utilisée. La Taq polymérase est une enzyme qui peut faire des erreurs en copiant l'ADN. Bien que la fréquence d'erreur de l'enzyme soit faible, ce phénomène peut aboutir à de mauvaise identification des populations, 2000.

. Spiegelman, La présence de certaines substances peut inhiber son activité et diminuer l'efficacité de l'amplification. Le résultat de cette inhibition se manifeste par des proportions entre les séquences d'ADN amplifiées non représentatives de l'échantillon initial. Les substances inhibitrices de l'enzyme sont entre autres, les acides humiques, les composés organiques, les polysaccharides?Toutes ces substances sont naturellement présentes dans l'échantillon initial et co-extraites avec l'ADN, La polymérase est également sensible aux conditions de réaction, 1997.

L. Biais-de-la, P. Sont-Également-dus-À-la-nature-de-l-'adn, and . Spiegelman, La PCR est une méthode durant laquelle se succèdent des cycles de dénaturation de l'ADN, d'hybridation des amorces et d'élongation de l'ADN. La dénaturation de l'ADN est réalisée par augmentation de la température. La température nécessaire pour dénaturer totalement un brin d'ADN est fonction de la composition en base du fragment. Pour les brins riches en bases GC, une température plus haute est nécessaire. Ainsi, la dénaturation est plus efficace pour les séquences riches en bases AT. La conséquence de ce phénomène est une amplification préférentielle des fragments riches en AT (Theron et Cloete Les proportions entre fragments d'ADN observées après l'amplification ne sont pas représentatives des proportions réelles, 2000.

. Acinas, Les chimères d'ADN correspondent à la recombinaison de fragments d'ADN sauvages proches. La séquence des deux fragments sauvages étant proche, ils forment par complémentarité un brin d'ADN chimère qui ne correspond à aucune population bactérienne, Ces brins chimères entraînent la sur-estimation de la diversité par la détection de faux positifs, 2005.

. Acinas, Des dimères d'amorces peuvent se former et biaiser la mesure de la diversité (Theron et Cloete, Ce phénomène peut également être observé pour les amorces, 2000.