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Human Genetics

Human Genetics. DNA Makes RNA Makes Protein. Terminology Review. Chromosome Threadlike structures in the nucleus that carry genetic information Gene Fundamental unit of heredity Inherited determinant of a phenotype Locus Position occupied by a gene on a chromosome  Gene

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Human Genetics

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  1. Human Genetics DNA Makes RNA Makes Protein

  2. Terminology Review Chromosome Threadlike structures in the nucleus that carry genetic information Gene Fundamental unit of heredity Inherited determinant of a phenotype Locus Position occupied by a gene on a chromosome  Gene sequence of DNA that instructs a cell to produce a particular protein DNA Deoxyribonucleic Acid-the molecule that forms genes The genetic material Allele Different DNA sequences possible for the same gene location

  3. “A genetic material must carry out two jobs: duplicate itself and control the development of the rest of the cell in a specific way.” -Francis Crick

  4. DNA (deoxyribonucleic acid) is a chain of nucleotides • Sugar: Deoxyribose • Phosphate • Base - one of four types: adenine (A), thymine (T) guanine (G), cytosine (C)

  5. Which of these are Purine bases?Pyrimidine bases? •  A (adenine) • C (cytosine) • T (thymidine) • G (guanine) • A guy walks into a bar and says "My name's Chargaff, and 22% of my DNA is "A" nucleotides. I'll bet anyone that they can't guess what percentage of my DNA is "C" nucleotides!" You say "I'm thirsty, so I'll take that bet!" http://escience.ws/b572/L1/L1.htm

  6. DNA Bases Pair through Hydrogen Bonds Erwin Chargaff observed: # of adenine = # of thymine # of guanine = # of cytosine Complementary bases pair: A and T pair C and G pair

  7. What's the difference between DNA and RNA?DNA contains the sugar deoxyribose while RNA is made with the sugar ribose. It's just a matter of a single 2' hydroxyl, which deoxyribose doesn't have, and ribose does have. Of course, you all remember that RNA uses the base uracil instead of thymine too.Cytosine naturally has a high rate of deamination to give uracil

  8. What's the difference between DNA and RNA? • DNA contains the sugar deoxyribose while RNA is made with the sugar ribose. It's just a matter of a single 2' hydroxyl, which deoxyribose doesn't have, and ribose does have. • You all remember that RNA uses the base uracil instead of thymine too. • Cytosine naturally has a high rate of deamination to give uracil

  9. If C-U deamination occurs and then is replicated, the U will pair with an A not the C with a G DEAMINATION ---------> Cytosine Uracil

  10. If 5-methyl C-T deamination occurs and then is replicated, the T will pair with an A not the C with a G DEAMINATION ---------> 5 methyl Cytosine Thymine

  11. DNA is a Double Helix • X-ray diffraction indicated DNA has a repeating structure. • Maurice Wilkins and Rosalind Franklin • DNA is double-stranded molecules wound in a double helix. • -James Watson and Francis Crick G C A T C G T A G C A T C G T A G C A T C G

  12. P A T P 5’ 3’ P C G P P P G C A P P P T P C G P P G C 3’ 5’ P P DNA Double Helix • A sugar and phosphate “backbone” connects nucleotides in a chain. • DNA has directionality. • Two nucleotide chains together wind into a helix • Hydrogen bonds between paired bases hold the two DNA strands together. • DNA strands are antiparallel

  13. Orientation of DNA The carbon atoms on the sugar ring are numbered for reference. The 5’ and 3’ hydroxyl groups (highlighted on the left) are used to attach phosphate groups. • The directionality of a DNA strand is due to the orientation of the phosphate-sugar backbone.

  14. Structure of DNA • Two nucleic acid chains running in opposite directions • The two nucleic acid chains are coiled around a central axis to form a double helix • For each chain – the backbone comes from linking the pentose sugar bases between nucleotides via phosphodiester bonds connecting via 3’ to 5’ • The bases face inward and pair in a highly specific fashion with bases in the other chain • A only with T, G only with C • Because of this pairing – each strand is complementary to the other 5’ ACGTC 3’ 3’ TGCAG 5’ •  Thus DNA is double stranded

  15. Chromatin = DNA and associated proteins DNA winds around histone proteins (nucleosomes). Other proteins wind DNA into more tightly packed form, the chromosome. Unwinding portions of the chromosome is important for mitosis, replication and making RNA.

  16. Genes: molecular definition • A gene is a segment of DNA • which directs the formation of RNA • which in turn directs formation of a protein • The protein (or functional RNA) creates the phenotype • Information is conveyed by the sequence of the nucleotides

  17. Why is DNA good Genetic Material? • A linear sequence of bases has a high storage capacity • a molecule of n bases has 4n combinations • just 10 nucleotides long -- 410 or 1,048,576 combinations • Humans – 3.2 x 109 nucleotides long – 3 billion base pairs

  18. Required properties of a genetic material • Chromosomal localization • Control protein synthesis • Replication

  19. DNA Replication • - the process of making new copies of the DNA molecules Potential mechanisms: organization of DNA strands Conservative old/old + new/new Semiconservative old/new + new/old Dispersive mixed old and new on each strand

  20. Meselson and Stahl’s replication experiment Conclusion: Replication is semiconservative.

  21. Replication as a process Double-stranded DNA unwinds. The junction of the unwound molecules is a replication fork. A new strand is formed by pairing complementary bases with the old strand. Two molecules are made. Each has one new and one old DNA strand.

  22. Fig 8.14

  23. Replication in vivo is complex • Replication requires the coordinated regulation of many enzymes and processes • unwind the DNA • synthesize a new nucleic acid polymer • proof read • repair mistakes

  24. Primase adds short primer to template strand Binding proteins stabilize separate strands Helicase unwinds parental double helix Ligase joins Okazaki fragments and seals other nicks in sugar-phosphate backbone Exonuclease removes RNA primer and inserts the correct bases DNA polymerase binds nucleotides to form new strands Enzymes in DNA replication

  25. 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ Binding proteins prevent single strands from rewinding. Primase protein makes a short segment of RNA complementary to the DNA, a primer. Replication Helicase protein binds to DNA sequences called origins and unwinds DNA strands.

  26. Overall direction of replication 3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ Replication DNA polymerase enzyme adds DNA nucleotides to the RNA primer. DNA polymerases require an underlying template (and a primer) and cannot synthesize in the direction 3' to 5'. That is, they cannot add nucleotides to a free 5' end.

  27. Overall direction of replication 3’ 5’ 3’ 5’ 3’ 3’ 5’ 5’ Replication DNA polymerase enzyme adds DNA nucleotides to the RNA primer. DNA polymerase proofreads bases added and replaces incorrect nucleotides.

  28. Overall direction of replication 3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ Replication Leading strand synthesis continues in a 5’ to 3’ direction.

  29. Overall direction of replication 3’ 3’ 5’ 5’ Okazaki fragment 3’ 5’ 3’ 5’ 5’ 3’ Replication Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.

  30. Replication Overall direction of replication 3’ 3’ 5’ 5’ Okazaki fragment 3’ 5’ 3’ 5’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.

  31. Replication 3’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.

  32. 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ Replication 3’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.

  33. 3’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ 5’ Replication Exonuclease enzymes remove RNA primers.

  34. 3’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 5’ Ligase forms bonds between sugar-phosphate backbone. Replication Exonuclease enzymes remove RNA primers.

  35. 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ 3’ 3’ 5’ 5’ 5’ 3’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ Replication

  36. General rules of conduct for DNA polymerase I enzymes (like Taq) • Remember your base pairing rules: G goes with C and A goes with T. • The 5' ends are strictly off limits • There will be no synthesis without a free 3' end • There will be no degradation without a free 3' end

  37. General rules of conduct for DNA polymerase I enzymes (like Taq) 5. There will be no synthesis without an underlying template 6. Under no circumstances may you make a synthetic addition to the 5‘ end 7. There is no reconstruction of a broken phosphodiester bond, unless you have ligase. If you are synthesizing DNA and run into an obstruction on your template, you must stop and leave the nick unrepaired.

  38. General rules of conduct for DNA polymerase I enzymes (like Taq) 8.If you have been provided with a free 3' end, a template, and a substrate molecule that is correct, you must add that nucleotide to the growing end of the strand (i.e. to the 3' end.)

  39. PCR: Polymerase Chain Reaction • Selective replication and amplification of specific(targeted) DNA sequences.  • PCR basics • Know some sequence of the piece of genomic or other DNA to be amplified • DNA primers - short DNA pieces of sequences complementary to the DNA sequence to be amplified • Four nucleotide building blocks • Taq1 - DNA polymerase, Buffer, MgCl2

  40. Polymerase Chain Reaction (PCR) DNA template is melted with high heat to separate strands. Denaturation Each DNA primer anneals, binding to its complementary sequence on the template DNA Annealing Extension DNA polymerase creates a new strand of DNA complementary to the template DNA starting from the primer’s free 3’ end. Multiple rounds of denaturation-annealing-extension are performed to create many copies of the template DNA between the two primer sequences.

  41. Polymerase Chain Reaction (PCR) DNA templateis denatured with heat to separate strands. 5’ 3’ G G A A C T A G C C T T G A T C G C 5’ 3’

  42. Polymerase Chain Reaction (PCR) DNA templateis melted with heat to separate strands. 5’ 3’ G G A A C T A G C C T T G A T C G C 5’ 3’

  43. A G T G C C 3’ A G T C A A Polymerase Chain Reaction (PCR) DNA polymerase creates a new strand of DNA complementary to the template DNA starting from the primer. 5’ 3’ G G A A C T A G C C T T 3’ 5’ 5’ G C G C T T G A T C G C 5’ 3’

  44. Polymerase Chain Reaction (PCR)

  45. Template Base Pairing • Requires correct temperature. • Too hot and nothing can form hydrogen bonds. • Too cold and the template reforms and the primers can form weak hydrogen bonds with sequences that are not perfectly complementary. • http://escience.ws/b572/L3/L3.htm

  46. Genome and Epigenome vary in Monozygotic Twins • Identical Twins don’t actually have completely identical DNA • http://www.cell.com/AJHG/abstract/S0002-9297(08)00102-X • Bruder et al. Phenotypically Concordant and Discordant Monozygotic Twins Display Different DNA Copy-Number-Variation Profiles AJHG, Vol 82, No, 3, 763-771 http://www.nytimes.com/2008/03/11/health/11real.html?scp=3&sq=epigenetics&st=cse

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