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Nathalie Smitz Introduction to molecular biology Chromosomes, carrier of heredity Chromosomes In Eukaryotic cells, chromosomes are isolated by a membrane within the nucleus. The number of chromosomes can vary a lot between species but also within it (karyotype). Human:46 chr Ant: 2 chr Butterfly: 440 chr Chromosomes In humans, there is two copies of each chromosome (1 coming from the mother and 1 coming from the father) = pairs of chromosomes. Ex. 23 pairs of chromosomes (22 pairs of nonsexual (autosomal) chromosomes and one pair of sex chromosomes) Chromosomes Each chromosome is made of a very long strand of DNA (desoxyribonucleic acid) that is highly compacted. Each DNA strand is a long double helix containing millions of steps. The steps of the helix consist of pairs of four types of molecules called nucleotides. DNA Watson & Crick proposed the double helix structure in 1953. DNA There are 4 nucleotide types : Adenine Guanine Thymine Cytosine Nucelotide complementarity: T/A and C/G: hydrogen bonding. DNA • Nucleotide succession forms genes that contains the genetic information used to synthesize proteins. Each chromosome contains hundreds to thousands genes (coding region) separated by non-coding regions. Purpose of DNA https://www.youtube.com/watch?v=zwibgNGe4aY Transfert of the genetic information DNA replication During replication, the double strand separates itself into two. Replication therefore assures the transfert of the genetic information from one cell to all its descendants during mitosis. https://www.youtube.com/watch?v=oebogqrX5F4 Meosis Mendel’s Principles of HeridityMendelian inheritance Johann Gregor Mendel Austrian monk (1822-1884), conducting hybridization experiments in garden peas (Pisum sativum); Cultivated and tested some 5,000 pea plants in the backyard of the church; Induced two generalizations which later became known as Mendel's Principles of Heredity or Mendelian inheritance, Experimental model Feature of the garden peas (Pisum sativum) : Self-fertilization Feature observable to the naked eye: seed shape, size, color, flower color, etc. Developed pure lineages (= homozygote), who will give descendants that will express unique feature Mendel laws First Law: Law of segregation When a plant with two dominant (DD) alleles is crossed with a plant having two recessive (rr) alleles, the first generation of plants will all have one dominant and one recessive (Dr) allele. smooth pea (DD) wrinkled pea (rr) Artificial crossfecondation Parents Cutted stamen before maturity D D r Dr Dr r Dr Dr 1st generation All peas are smooth, no wrinkled pea Mendel laws Autofecondation of 1st generation individuals (phenotype: smooth) Wrinkled pea appears at second generation; Wrinkled feature is carried by the 1st generation but not expressed ; In the second generation, on average one of four plants will have two recessive alleles. D r D DD Dr r Dr rr Mendel laws Second law: Law of independent assortment When parents express multiple features- ex. shape (smooth vs wrinkled) and color (green vs yellow), the features are independently transmitted each from another. The hybrids of first generation F1 express exclusively dominant features (here, yellow and smooth) . At second generation, descendants will express all possible combination between those features. Heredity Mendel wasn’t speaking about “genes” but about transmission of some features through generations. In 1900, three botanists, De Vries, Correns and Von Tschermak, performed similar experimentations with other species model and obtained the same results. The word “gene” was invented to define the transmission of features as supposed by Mendel no link yet with chromosomes. Heredity Mendel laws were forgotten for more than 35 years, until Morgan demonstrated that the color of fruit flies' eyes was transmitted differently between males and females. Morgan elaborated a theory of heredity linked to the gender: the gene responsible of the color of the eyes in fruit flies would be located on the X chromosome. He therefore propose that the genetic information may be supported by the chromosomes. Main techniques of molecular biology PCR PCR or « polymerase chain reaction » was invented by Kary Mullis, which obtained a Nobel price in 1993 for this discovery. Important discovery because it allows to amplify specific targeted region of the genome. It evolved during the last decades but the original principle is relatively easy. PCR Exponential reaction with two principal operators : The Taq Polymerase, an enzyme resistant to high temperature, originally isolated from the thermophile bacteria Thermus aquaticus, that can be found nearby hot water springs. The dNTPs (DesoxyNucleotides-Tri- Phosphates) are molecules containing a nucelotide bounds to three phosphates. These are the basic elements used by the Taq Polymerase to synthetise complementary DNA strand. PCR There are three steps : Denaturation : separation of the two DNA strands through an increase of the temperature; Annealing of the primers : after temperature decrease, the specific primer complementary to the DNA strand will hybredize to the region to amplify; Elongation : the synthesis of the complementary strand will be initiated: the Taq polymerase will add the complementary dNTPs, which are present in the reaction solution. PCR Different temperature for each steps: PCR The PCR is an exponential reaction that uses each produced product from one step as matrices for the next step. This process will produce thousands of copies of a targeted region of the genome Practically, the thermocycler is the device that is used to perform PCR by changing of temperature cyclically. DNA sequencing This technic will allow to reveal which is the nucelotide succession of the targeted genome region. The most used technic is the SANGER sequencing: Similar to PCR, the difference is that dNTP are mixed with ddNTP (didésoxyribonucléotides): the Taq polymerase is blocked and the DNA strand synthesis is stopped when a ddNTP is inserted during synthesis. Get an assembly of DNA amplicons of variable size, depending on the position where the ddNTP was inserted. DNA sequencing DNA sequencing This technic is sometimes replaced by « Next Generation Sequencing », which allows to obtain larger fragments of the genome, but : More expensive; Analyses complexity; … Some molecular phylogenic concepts Molecular phylogeny Molecular phylogeny allows to retrace the evolutionary history of species through the study of the evolution of mutations within specific gene(s), using statistical methods. To compare obtained sequences, they must be first aligned. Ex: MEGA, a freely accessible software, allows to do such aligments. Molecular phylogeny After alignment, mutations can be identified: Transitions between purines or between pyrimidines Transversions from a purine toward a pyrimidine Deletions or insertions A C G T Molecular phylogeny Different alignment methods exist and the choice depends of the data to analyse and the type of analyses that will be performed. Some sequences are freely accessible on online repository as for example on GENBANK. http://www.ncbi.nlm.nih.gov/genbank/ Molecular phylogeny Then after, it is possible to reconstruct phylogenetic trees, associating first the sequences that are the most similar, toward the most different one. For this recontruction, we first need to identify the evolutionay model that best fit the database. It will evaluate: The evolution of one sequence in comparision to its ancestral sequence; The divergence between the two sequences; The mutation frequency; Base frequency; Substitution rate etc Molecular phylogeny The most complex evolutionary model it the GTR; Different results can be obtained if using different evolutionary model importance in choosing! Two different phylogenetics trees obtained with the same initial database but using distinct evolutionary model. Molecular phylogeny When the appropriate model is selected, there are still some other parameters to define; Different software are needed to buil the different trees, depending on the methodology (Neighbour joining, bayesian analysis, maximum likelihood, maximum parsimony) Different ways to recontruct a phylogenetic tree For more information, please refer to the ‘phylogenetic handbook’ Molecular phylogeny The best is to perform the tree recontructions with the different technics and compare the obtained results between each of them. To have a first idea of the tree structure, you can already do one with the MEGA software used for the alignment. DNA Barcoding DNA Barcoding DNA Barcoding is a technic that allows the identification of specimen using a short section of DNA from a standardized region of the genome A good DNA barcode gene is : A variable sequence between specis but conserved within species, which confers her a high discrimination power. A relatively short sequence sothat it may be sequenced but suffently long to have enough informative sites. Commonly, it is the COI gene that is used for this purpose- of about 650base pairs, at least in metazoans. DNA Barcoding DNA barcoding is used for different purposes: Identification of cryptic species (similar morphologies difficulties of morphological identification) Identification of incomplete specimens Identification at large scale to study the biodiversity and the environmental risks Identification of invasive species Identification of new species Association of males and femelles from a same species (sexual dimorphism) Association of different stades of development within a same species … Etc. DNA Barcoding Confirmation of morphological studies e.g. revision of Hepsetus DNA Barcoding But taxonomic analyses cannot be replaced by barcoding! DNA barcoding can help and facilitate the processus of identification and allow to discover new species or answer to other biological questions BUT cannot replace classical taxonomic techniques complementarity! Ex. integrative studies. DNA Barcoding Molecular barcoding is a campaign largely financed in the world, with different organisations : « Barcode of Life Data Systems » (BOLD) : www.boldsystems.org « Consortium for Barcode of Life » (CBOL) : http://www.barcodeoflife.org/ « European Consortium for the Barcode of Life »: http://www.ecbol.org/ « international Barcode of Life » (iBOL): www.iBOL.org « The Belgian Network for DNA Barcoding » (BeBOL) : http://bebol.myspecies.info/ « Canadian Centre for DNA Barcoding » (CCDB) : http://www.ccdb.ca/ « The Fish Barcode of Life Initiative » (Fish-BOL) : http://www.fishbol.org/ …. DNA Barcoding But practicaly, its not so easy… « Universal » primer described in the litterature are not adapted to all organisms : additional adjustments are necessary. Questions?