DNA: The Secret Of Our Life(An Introduction Lecture)lecture 1
Lecture Objectives• At the of this Lecture you should be able tolearn:• 1. The history of DNA discovery with theirimportance for the new concepts andapplication techniques• 2. The chemical and physical properties ofDNA and how these properties wereexploited for DNA technologies
Early historical perspective
We enter the 20thcentury with anunderstanding of the DNA buildingblock.
Some Experimental Data leading toDNA as biological source of DNA• Griffith’s• Avery et al.• Hershey and Chase• Chargaff• Wilkins and Franklin• Watson and Crick
Back to Franklin and Wilkins Data: Pairing of specific classes ofbases can account for diameter of DNAJust right!6 sided ring6 sided ring +5 sided ring
Most Common Secondary Structure (3D structure)• B-DNA• Alpha Helix• Right Handed Turn• 10 bases per 360º turn
A function of Major and Minor Grooves
23Nucleosides• Nucleosides: nitrogenous base linked to specific sugar– RNA: adenosine, guanosine, cytidine, uridine– DNA: deoxyadenosine, deoxyguanosine,deoxycytidine, (deoxy)thymidine22.214.171.124/.../Nucleosides.gifDNA nucleoside RNA nucleoside
24NucleotidesThe nucleotide structure consists of– the nitrogenous base attached to the 1’ carbonof deoxyribose– the phosphate group attached to the 5’ carbonof deoxyribose– a free hydroxyl group (-OH) at the 3’ carbon ofdeoxyribose
26DNA StructureNucleotides are connected to each other toform a long chainphosphodiester bond: bond betweenadjacent nucleotides– formed between the phosphate group of onenucleotide and the 3’ –OH of the nextnucleotideThe chain of nucleotides has a 5’ to 3’orientation.
28DNA structure determinationChargaffs Rules– Erwin Chargaff determined that• amount of adenine = amount of thymine• amount of cytosine = amount of guanine
29DNA StructureThe double helix consists of:– 2 sugar-phosphate backbones– nitrogenous bases toward the interior of themolecule– bases form hydrogen bonds with complementarybases on the opposite sugar-phosphate backbone• Adenine pairs with Thymine (2 H bonds)• Cytosine pairs with Guanine (3 H Bonds)
31DNA StructureThe two strands of nucleotides areantiparallel to each other– one is oriented 5’ to 3’, the other 3’ to 5’The two strands wrap around each other tocreate the helical shape of the molecule.
33Chemical Properties of DNA• Factors that affect DNA structure– Temperature: denaturation (can be reversible)– pH: high pH can denature DNA– Salt concentration: lowering salt concentrationcan denature DNA– Molecular Hybridization (DNA:DNA) and(DNA:RNA)– UV absorption (230-260nm)
• Southern blotting of DNA fragmentsAPPLICATION Researchers can detect specific nucleotide sequences within a DNA sample with this method. Inparticular, Southern blotting is useful for comparing the restriction fragments produced fromdifferent samples of genomic DNA.TECHNIQUE In this example, we compare genomic DNA samples from three individuals: a homozygotefor the normal -globin allele (I), a homozygote for the mutant sickle-cell allele (II), and aheterozygote (III).DNA + restriction enzyme Restrictionfragments I II IIII Normal-globinalleleII Sickle-cellalleleIII HeterozygotePreparation of restriction fragments. Gel electrophoresis. Blotting.GelSpongeAlkalinesolutionNitrocellulosepaper (blot)HeavyweightPapertowels1 2 3Figure 20.10
RESULTS Because the band patterns for the three samples are clearly different, this method can be used toidentify heterozygous carriers of the sickle-cell allele (III), as well as those with the disease, who havetwo mutant alleles (II), and unaffected individuals, who have two normal alleles (I). The band patternsfor samples I and II resemble those observed for the purified normal and mutant alleles, respectively,seen in Figure 20.9b. The band pattern for the sample from the heterozygote (III) is a combinationof the patterns for the two homozygotes (I and II).Radioactivelylabeled probefor -globingene is addedto solution ina plastic bagProbe hydrogen-bonds to fragmentscontaining normalor mutant -globinFragment fromsickle-cell-globin alleleFragment fromnormal -globinallelePaper blotFilm overpaper blotHybridization with radioactive probe. Autoradiography.I II IIII II III1 2
• DNA microarray assay of gene expression levelsAPPLICATIONTECHNIQUETissue samplemRNA moleculesLabeled cDNA molecules(single strands)DNAmicroarraySize of an actualDNA microarraywith all the genesof yeast (6,400spots)Isolate mRNA.1With this method, researchers can test thousands of genes simultaneously to determinewhich ones are expressed in a particular tissue, under different environmental conditions in various diseasestates, or at different developmental stages. They can also look for coordinated gene expression.Make cDNA by reverse transcription, using fluores-cently labeled nucleotides.2Apply the cDNA mixture to a microarray, a microscope slide on which copies of single-strandedDNA fragments from the organism‘s genes are fixed, a different gene in each spot. The cDNAhybridizes with any complementary DNA on the microarray.3Rinse off excess cDNA; scan microarray for fluorescence. Each fluorescent spot(yellow) represents a gene expressed in the tissue sample.4RESULTThe intensity of fluorescence at each spot is a measure of the expression of the generepresented by that spot in the tissue sample. Commonly, two different samples are tested together bylabeling the cDNAs prepared from each sample with a differently colored fluorescence label. Theresulting color at a spot reveals the relative levels of expression of a particular gene in the two samples,which may be from different tissues or the same tissue under different conditions.Figure 20.14