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From DNA To RNA To Protein

From DNA To RNA To Protein. OH. Phosphate. NH 2. P. HO. O. Base. O. N. N. N. N. CH 2. O. Sugar. H. OH. OH. A Nucleotide. NH 2. Thymine (DNA). Uracil (RNA). CH 3. Adenine. N. N. N. N. N. N. NH. N. N. NH. N. O. N. O. O. O. NH. O. O. N. N. O. NH 2.

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From DNA To RNA To Protein

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  1. From DNATo RNATo Protein

  2. OH Phosphate NH2 P HO O Base O N N N N CH2 O Sugar H OH OH A Nucleotide

  3. NH2 Thymine (DNA) Uracil (RNA) CH3 Adenine N N N N N N NH N N NH N O N O O O NH O O N N O NH2 NH2 Guanine Cytosine NH2 N N Two Families of Bases Purines Pyrimidines

  4. Hydrogen bonds 2 H- bonds for A:T 3 H-bonds for G:C

  5. Deoxyribose Ribose

  6. Genomes vary in size

  7. DNA Transcription Ribosome mRNA Translation Polypeptide (protein) IntroductionThe Central Dogma of Molecular Biology Cell

  8. 5’ 3’ 3’ 5’ RNA Pol. Ribosome mRNA 5’ Transcription And Translation In Prokaryotes

  9. Cytoplasm Nuclear pores Ribosome AAAAAA AAAAAA DNA Transcription Translation preRNA RNA Processing G G mRNA Export Nucleus Eukaryotic Transcription

  10. Nucleotide Words • Words in the nucleotide language are all 3 letters or bases long. • These three base “words” are called codons • This means that there can only be 43 = 64 unique words.

  11. OH NH2 P HO O Adenine N N O N N CH2 O B A S E S H O O Guanine P HO O N NH O SUGAR-PHOSPHATE BACKBONE N NH2 N CH2 O Arginine H O NH2 Adenine P HO O N N O N N CH2 O OH H A Codon

  12. Redundancy in the Code • Codons code for only 20 words, or amino acids. • The fact that many amino acids are coded for by several codons is called degeneracy

  13. The Genetic Code

  14. Methionine A C C 73 1 72 2 71 3 70 4 69 5 68 6 67 59 7 66 Py A* U* 65 64 63 62 C 16 Pu 17 9 A Pu 17:1 13 12 Py 10 49 50 51 52 G C T y G* Py 22 23 Pu 25 47:16 G A 26 47:15 20 20:2 20:1 27 1 43 44 28 42 45 46 29 41 47 30 40 47:1 31 39 Py* 38 U Pu* U 34 36 C 35 A Anticodon Met-tRNA

  15. fMet P A Large subunit E UAC 5’ GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 3’ mRNA Small subunit Translation - Initiation

  16. Polypeptide Arg Met Phe Leu Ser Aminoacyl tRNA Gly P A UCU Ribosome E CCA 5’ GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 3’ mRNA Translation - Elongation

  17. סוגי RNAבתא RNA .1ריבוזומלי rRNA 80% 10% 2 . RNAנשא tRNA 3. RNAשליח mRNA 5% 4. אחרים snRNA , gRNA 5%

  18. Genetic engineer method: • Restriction Enzymes • PCR

  19. Restriction Enzymes (REs) are endonucleases which cut ONLY double-stranded DNA that contain a particular nucleotide sequence (recognition site) ALWAYS in the same way • Bacterial enzymes, destroy the foreign DNA

  20. 5' - G A A T T C - 3' 3' - C T T A A G - 5' EcoRI Most REs recognise PALINDROMIC sequences The sequence on one strand reads the same in the opposite direction on the complementary strand. GTAATG is not a palindromic DNA sequence

  21. Potential "restriction sites" appear in almost anygenethat can snip it out. The sequences of some artificialplasmids include a" linker" that contains dozens of restriction enzyme recognition sequences within a very short segment of DNA. Restriction Enzyme/s Application of REs Gene cloning

  22. Gene cloning REs will produce ends that enable the gene to be spliced into a plasmid Ligation

  23. PCR • Inventor: 1983 Kary Mullis • Nobel prize in chemistry in 1993 needs only slightly DNA molecules to produce a huge range of copies PCR needs unleast some information of the gene order (or from some similar gene) to make the primer

  24. Tools for PCR A small amount of DNA Taq DNA Polymerase (or another thermally stable DNA polymerase) Nucleotides Primers • Two different kind of • Usually about 20 nucleotides

  25. PCR Melting 100 94 oC Temperature 50 0 T i m e 3’ 5’ 5’ 3’

  26. PCR Melting 100 94 oC Temperature 50 0 T i m e 3’ 5’ Heat 5’ 3’

  27. PCR Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 0 T i m e 5’ 3’ 5’ 5’ 5’ 3’

  28. PCR 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 0 T i m e 5’ 3’ 5’ 5’ 5’ 3’ Heat Heat 5’

  29. PCR 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 0 T i m e 5’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’

  30. PCR 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 3’ 5’ 0 5’ T i m e 5’ 5’ 3’ 5’ 5’ 5’ Fragments of defined length 5’ 5’ 5’ 5’ 5’

  31. DNA Between The Primers Doubles With Each Thermal Cycle Number 1 2 4 8 16 32 64 0 Cycles 1 2 3 4 5 6

  32. PCR Program Denaturation 95oC (30-60s) Annealing (1-2min.) Elongation 72oC x20-30cycles Initial denaturation 95oC (3-5min) Prior to the first cycle, the DNA is often denatured for an extended time to ensure that both the template DNA and the primers have completely separated and are now single-strand only. Also certain polymerases are activated at this step (hot-start PCR). Final extension (10min) To ensure that any remaining single stranded DNA is completely copied.

  33. Identification of PCR product

  34. Identification of PCR product

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