Dna in basic
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The document provides a summary of the history and discovery of DNA. It discusses early experiments in the 1920s-1950s that provided evidence DNA was the genetic material, including Griffith's experiment, Avery's work, and Hershey and Chase's experiment. It then summarizes Watson and Crick's pivotal discovery in 1953 of the double helix structure of DNA, for which they received the Nobel Prize in 1962. The document outlines the chemical and physical properties of DNA including its nucleotides, base pairing, and double helix structure.
Dna in basic
- 1. DNA: The Secret Of Our Life (An Introduction Lecture) lecture 1
- 2. Lecture Objectives • At the of this Lecture you should be able to learn: • 1. The history of DNA discovery with their importance for the new concepts and application techniques • 2. The chemical and physical properties of DNA and how these properties were exploited for DNA technologies
- 4. We enter the 20th century with an understanding of the DNA building block.
- 5. Some Experimental Data leading to DNA as biological source of DNA • Griffith’s • Avery et al. • Hershey and Chase • Chargaff • Wilkins and Franklin • Watson and Crick
- 6. Griffith’s Experiment: 1928 Conclusion: A Transformation “factor” exists
- 7. Support for nucleic acid transfer Hershey and Chase Experiment, 1952: Confirms DNA as genetic material
- 8. Conclusion: DNA identified as source of genetic information
- 9. Franklin and Wilkins 1947 1920-1958 1916-2004
- 11. Watson and Crick, 1953 inferred the DNA structure
- 12. Nobel Prize: 1962 1928-1916-2004 1916-2004
- 13. The building blocks of DNA are nucleotides.
- 15. RNA’s Sugar DNA’s Sugar
- 17. DNA nucleotides
- 19. Back to Franklin and Wilkins Data: Pairing of specific classes of bases can account for diameter of DNA Just right! 6 sided ring 6 sided ring + 5 sided ring
- 21. Most Common Secondary Structure (3D structure) • B-DNA • Alpha Helix • Right Handed Turn • 10 bases per 360º turn
- 22. A function of Major and Minor Grooves
- 23. 23 Nucleosides• Nucleosides: nitrogenous base linked to specific sugar – RNA: adenosine, guanosine, cytidine, uridine – DNA: deoxyadenosine, deoxyguanosine, deoxycytidine, (deoxy)thymidine 138.192.68.68/.../Nucleosides.gif DNA nucleoside RNA nucleoside
- 24. 24 Nucleotides The nucleotide structure consists of – the nitrogenous base attached to the 1’ carbon of deoxyribose – the phosphate group attached to the 5’ carbon of deoxyribose – a free hydroxyl group (-OH) at the 3’ carbon of deoxyribose
- 25. 25 Nucleotides • Subunits of DNA and RNA – Nucleosides linked to phosphate group via ester bond – “dNTP’s”: DNA – “rNTP’s”: RNA
- 26. 26 DNA Structure Nucleotides are connected to each other to form a long chain phosphodiester bond: bond between adjacent nucleotides – formed between the phosphate group of one nucleotide and the 3’ –OH of the next nucleotide The chain of nucleotides has a 5’ to 3’ orientation.
- 27. 27
- 28. 28 DNA structure determination Chargaff's Rules – Erwin Chargaff determined that • amount of adenine = amount of thymine • amount of cytosine = amount of guanine
- 29. 29 DNA Structure The double helix consists of: – 2 sugar-phosphate backbones – nitrogenous bases toward the interior of the molecule – bases form hydrogen bonds with complementary bases on the opposite sugar-phosphate backbone • Adenine pairs with Thymine (2 H bonds) • Cytosine pairs with Guanine (3 H Bonds)
- 30. 30
- 31. 31 DNA Structure The two strands of nucleotides are antiparallel to each other – one is oriented 5’ to 3’, the other 3’ to 5’ The two strands wrap around each other to create the helical shape of the molecule.
- 32. 32
- 33. 33 Chemical Properties of DNA • Factors that affect DNA structure – Temperature: denaturation (can be reversible) – pH: high pH can denature DNA – Salt concentration: lowering salt concentration can denature DNA – Molecular Hybridization (DNA:DNA) and (DNA:RNA) – UV absorption (230-260nm)
- 34. • Southern blotting of DNA fragments APPLICATION Researchers can detect specific nucleotide sequences within a DNA sample with this method. In particular, Southern blotting is useful for comparing the restriction fragments produced from different samples of genomic DNA. TECHNIQUE In this example, we compare genomic DNA samples from three individuals: a homozygote for the normal -globin allele (I), a homozygote for the mutant sickle-cell allele (II), and a heterozygote (III). DNA + restriction enzyme Restriction fragments I II III I Normal -globin allele II Sickle-cell allele III Heterozygote Preparation of restriction fragments. Gel electrophoresis. Blotting. Gel Sponge Alkaline solution Nitrocellulose paper (blot) Heavy weight Paper towels 1 2 3 Figure 20.10
- 35. RESULTS Because the band patterns for the three samples are clearly different, this method can be used to identify heterozygous carriers of the sickle-cell allele (III), as well as those with the disease, who have two mutant alleles (II), and unaffected individuals, who have two normal alleles (I). The band patterns for 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 combination of the patterns for the two homozygotes (I and II). Radioactively labeled probe for -globin gene is added to solution in a plastic bag Probe hydrogen- bonds to fragments containing normal or mutant -globin Fragment from sickle-cell -globin allele Fragment from normal -globin allele Paper blot Film over paper blot Hybridization with radioactive probe. Autoradiography. I II III I II III 1 2
- 36. • DNA microarray assay of gene expression levels APPLICATION TECHNIQUE Tissue sample mRNA molecules Labeled cDNA molecules (single strands) DNA microarray Size of an actual DNA microarray with all the genes of yeast (6,400 spots) Isolate mRNA.1 With this method, researchers can test thousands of genes simultaneously to determine which ones are expressed in a particular tissue, under different environmental conditions in various disease states, or at different developmental stages. They can also look for coordinated gene expression. Make cDNA by reverse transcription, using fluores-cently labeled nucleotides.2 Apply the cDNA mixture to a microarray, a microscope slide on which copies of single-stranded DNA fragments from the organism‘s genes are fixed, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray. 3 Rinse off excess cDNA; scan microarray for fluorescence. Each fluorescent spot (yellow) represents a gene expressed in the tissue sample. 4 RESULT The intensity of fluorescence at each spot is a measure of the expression of the gene represented by that spot in the tissue sample. Commonly, two different samples are tested together by labeling the cDNAs prepared from each sample with a differently colored fluorescence label. The resulting 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