Introduction to Molecular Biology PDF

INTRODUCTION TO MOLECULAR BIOLOGY Definition Molecular biology is the study of gene structure and function at the molecular level What is a gene? The question was asked by three groups: The chemists question: of what (which cellular chemical) is the gene made?...

INTRODUCTION TO MOLECULAR BIOLOGY Definition Molecular biology is the study of gene structure and function at the molecular level What is a gene? The question was asked by three groups: The chemists question: of what (which cellular chemical) is the gene made? The geneticists question: what does the gene do? The physicists question: what are the thermodynamics of the gene? CHEMICAL COMPONENTS OF THE NUCLEIC ACIDS prime base Nitrogenous bases no e nitrogen Phosphoric acid Pentoses missing -  NUCLEOSIDES Glycoside bond between the Nitrogenous base and the Pentose: Nucleoside. base withsugar Bond forms between the hydroxyl group (OH) at C1 of sugar the pentose and the Hydrogen (H) at position H1 (pyrimidine) or H9 (purine). > - foro  LES NUCLEOTIDES I Addition of the phosphate group at position C5’ Nucleotides 0 do not forget this d Part an Beta secon I DNA molecule is acidic due to the phosphate goup. At physiological pH, the phosphate dissociate and become negatively charged with the release of an H+. Starting from the carbone 5’, phosphate are called alpha, beta and gamma.  3 PHOSPHODIESTER BONDS s prime Antiparallel s I Nucleic acidsF(DNA and RNA) are formed nucleotides polymerization. This reaction is catalysed by polymerases. Iitinhemde.IE The bond that form between two nucleotides is called phosphodiester enin bond. These bonds form between carbone at position 3’ of one sugar and the carbone at position 5’ of the next sugar. y 5 I L Sugar Phosphodiester bond o 1 2  Example of simplified DNA extraction procedure No need of microscope to see DNA. All you need is cooking recipe. HI Materiel needed 1 large onion, picture Salt Lemon juice, Report alcool 95%,919 ethanol net Recipe 1) Cut the onion into large piece, add few milliliter of water and smash using a mixer. 2) Filter the mixture through a net and add to the filtred solution salt and lemon juice. Mix well. 3) Add three volumes of ethanol. Mix gently and put in the freezer to help the precipitation of the nucleic acid. Representing the DNA double helix o do in gi parallel causeit'sAnti  PROPERTIES OF THE DOUBLE HELIX The 2 DNA strand possess 3 major properties: theDNAAntiparallel why nitrogenbases foldbetween ABand orV - Complementary - Antiparallele sieaiii - Helicoidal I  COMPLEMENTARY STRANDS The energy of the hydrogen bonds between complementary strands represents the force that hold the strands together. Precentage whythe different C of Gslightly G thanthe P of Aadt a For sterical reasons, purine is facing pyrimidine base and vice-versa. Edwin Chargaff ,in 1951, demonstrated that the 4 nucleotides are not equally present in the DNA and that the ratio varies a lot between species. A T ANTIPARALLELE POLARITY OF THE POLYNUCLEOTIDE CHAINE 5’carbon 5’ 3’ 3’carbon 3’ 5’  HELICOIDAL In a DNA molecule, the two strands are not parallel, but intertwined with each other. Each strand looks like a helix. The two strands form a "double helix" structure, which was first discovered by James D. Watson and Francis Crick in 1953. The double helix can be found under three different form. Properties of the helix Form A Form B Form Z Hybrid RNA-DNA Sens of the helix Right Right Left Right rotation Residus per turn 11 10 12 11 Rotation per 33 deg. 36 deg. –30 deg. 33 deg. residu Distance between 0.255 nm 0.34 nm 0.37 nm 0.255 nm bases step 2.8 nm 3.4 nm 4.5 nm 2.8 nm  Form A Form B A form appears when the amount of water in the media is around 75%. B form appears when the amount of water in the media is around 92%. Z form is seen in the GC-rich regions DNA-RNA hybrid has an A form.  MAJOR AND MINOR GROOVE IN THE B FORM DNA MOLECULE The intertwined strands make two grooves of different widths, referred to as the major groove and the minor groove, which may facilitate binding with specific proteins.  HYPERCHROMY Absorbance at 260 nm of DNA solution increase when the double helix is denatured into single strand. Single strand Absorbance Double strand gnawa a it Its Ifd ifeng.int 220 260 300 temprture danger very DNA's hyperchromic effect means that ssDNA absorbs more UV than does dsDNA. The absorbance for single-stranded DNA will be 40% higher than that for double stranded DNA at the same concentration.  TEMPERATURE OF FUSION (Tm) or melting point refold protiencannot The Tm (temperature of fusion or melting temperature) of a given DNA sequence will correspond to the temperature at which 50% of the DNA is single strand. The Tm depends of the length, composition of the DNA molecule and the presence of ions in the media. oimt theftp.sfgigdatgai  THE Tm IS DEPENDENT ON THE CONTENT IN GC IN THE DNA MOLECULE The.IE ftingpoint The average GC content of a given DNA molecule can be determined from the Tm of this molecule. Tm= 4x[GC] + 2x [AT] staff Tm below the Tmthey Acsosated REASSOCIATION KINETIC 31 AskMouza  PARTIALLY DENATURED DNA OBSERVED UNDER ELECTRONIC MICROSCOPE Double strand, G-C rich region (DNA not yet denatured) closed is Area G C Denatured A-T rich region. D axis Formation of a single strand containing bubble A-T rich region separates first and then the GC rich region. MOLECULAR HYBRIDIZATION  NUCLEIC ACID HYBRIDIZATION Fawn's DNA replication and transcription requires temporarily separation of the two strands. The separation is catalyzed by different enzymatic activities. In vitro, such separation can also be achieved by heating the DNA. The heat energy increases the movement of the molecules which breaks the hydrogen bonds to the separation of the two strand. The two strands within a DNA molecule can also be separated chemically by basic solutions such as NaOH or by solutions containing high concentration of urea or formamide. The separated strand will adopt a random coiling conformation. By decreasing progressively the temperature, the double helix will form again. This phenomenon is called HYBRIDIZATION. viii we heattheDNA in95 CREATION OF DNA-RNA HYBRID. Hybridization can occur either: thecool prevent atom 1- in solution 2- on solid support : immobilization on membranes, on bacteria colonies, chromosomes and tissues section…