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DNA, Genes, and Biotechnology

DNA, Genes, and Biotechnology. Chapter 22. Structure of the Hereditary Material. Experiments in the 1950s showed that DNA is the hereditary material Scientists raced to determine the structure of DNA 1953 - Watson and Crick proposed that DNA is a double helix.

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DNA, Genes, and Biotechnology

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  1. DNA, Genes, and Biotechnology Chapter 22

  2. Structure of the Hereditary Material • Experiments in the 1950s showed that DNA is the hereditary material • Scientists raced to determine the structure of DNA • 1953 - Watson and Crick proposed that DNA is a double helix

  3. Structure of Nucleotides in DNA • Each nucleotide consists of • Deoxyribose (5-carbon sugar) • Phosphate group • A nitrogen-containing base • Four bases • Adenine, Guanine, Thymine, Cytosine (A) (G) (T) (C)

  4. Nucleotide Bases ADENINE (A) GUANINE (G) phosphate group deoxyribose THYMINE (T) CYTOSINE (C)

  5. Watson-Crick Model • DNA consists of two nucleotide strands • Strands run in opposite directions • Strands are held together by hydrogen bonds between bases • A binds with T, C with G • Molecule is a double helix

  6. Structure of DNA A gene is a sequence of nucleotides in a DNA molecule

  7. DNA Structure Helps Explain How It Duplicates • DNA is two nucleotide strands held together by hydrogen bonds • Hydrogen bonds between two strands are easily broken • Each single strand then serves as template for new strand

  8. DNA Replication • Each parent strand remains intact • Every DNA molecule is half “old” and half “new” new old old new

  9. Base-Pairing during Replication Each old strand serves as the template for complementary new strand

  10. Errors in DNA Replication • Mistakes can occur during replication • Enzymes may detect and correct the problem, restoring the proper DNA sequence • When the error is not corrected, the result is a mutation

  11. Gene Mutations Base-Pair Substitutions Insertions Deletions

  12. Effect of Base-Pair Substitution original base triplet in a DNA strand a base substitution within the triplet (red) As DNA is replicated, proofreading enzymes detect the mistake and make a substitution for it: POSSIBLE OUTCOMES: OR One DNA molecule carries the original, unmutated sequence The other DNA molecule carries a gene mutation

  13. Frameshift Mutations • Insertion • Extra base added into gene region • Deletion • Base removed from gene region • Both shift the reading frame • Result in many wrong amino acids

  14. Frameshift Mutation mRNA PARENTAL DNA amino acid sequence ARGININE GLYCINE TYROSINE TRYPTOPHAN ASPARAGINE altered mRNA BASE INSERTION altered amino-acid sequence ARGININE GLYCINE LEUCINE LEUCINE GLUTAMATE

  15. Transposable Elements • DNA segments that move spontaneously about the genome • When they insert into a gene region, they usually inactivate that gene • Neurofibromatosis

  16. DNA to RNA to Proteins transcription translation protein RNA DNA

  17. Three Classes of RNAs • Messenger RNA • Carries protein-building instruction • Ribosomal RNA • Major component of ribosomes • Transfer RNA • Delivers amino acids to ribosomes

  18. Base-Pairing during Transcription • A new RNA strand can be put together on a DNA region DNA G C A U G C A T RNA C G T A C G T A DNA DNA base-pairing in DNA replication base-pairing in transcription

  19. Promoter • A base sequence in the DNA that signals the start of a gene • For transcription to occur, RNA polymerase must first bind to a promoter

  20. Gene Transcription DNA to be transcribed unwinds transcribed DNA winds up again mRNA transcript RNA polymerase

  21. snipped out snipped out Transcript Modification unit of transcription in a DNA strand 3’ 5’ exon intron exon intron exon transcription into pre-mRNA poly-A tail cap 5’ 3’ 5’ 3’ mature mRNA transcript

  22. Gene Regulation • Most cells of your body carry the same genes • Each uses only a tiny subset at any time, and some are never turned on • Regulatory proteins can speed up or halt transcription

  23. Genetic Code • Set of 64 base triplets • Codons • Nucleotide bases read in blocks of three • 61 specify amino acids • 3 stop translation

  24. Code Is Redundant • Twenty kinds of amino acids are specified by 61 codons • Most amino acids can be specified by more than one codon • Six codons specify leucine • UUA, UUG, CUU, CUC, CUA, CUG

  25. tRNA Structure codon in mRNA anticodon in tRNA tRNA molecule’s attachment site for amino acid amino acid OH

  26. Ribosomes tunnel small ribosomal subunit large ribosomal subunit intact ribosome

  27. Three Stages of Translation Initiation Elongation Termination

  28. Initiation • Initiator tRNA binds to small ribosomal subunit • Small subunit/tRNA complex attaches to mRNA and moves along it to an AUG “start” codon • Large ribosomal subunit joins complex

  29. Binding Sites on Large Subunit binding site for mRNA A (second binding site for tRNA) P (first binding site for tRNA)

  30. Elongation

  31. Termination • A stop codon in the mRNA moves onto the ribosomal binding site • No tRNA has a corresponding anticodon • Proteins called release factors bind to the ribosome • mRNA and polypeptide are released

  32. Making Recombinant DNA Restriction enzymes cut DNA into fragments 5’ G A A T T C 3’ C T T A A G one DNA fragment another DNA fragment 5’ G A A T T C 3’ 5’ C T T A A G 3’ Fragments base-pair at sticky ends

  33. Using Plasmids • Plasmid is small circle of bacterial DNA • Foreign DNA can be inserted into plasmid • Forms recombinant plasmids • Plasmid is a cloning vector • Can be used to deliver DNA into another cell

  34. Using Plasmids DNA fragments + enzymes recombinant plasmids host cells containing recombinant plasmids

  35. Polymerase Chain Reaction • Sequence to be copied is heated • Primers are added and bind to ends of single strands • DNA polymerase uses free nucleotides to create complementary strands • Doubles number of copies of DNA

  36. double-stranded DNA to copy PCR (1) DNA heated to 90°– 94°C primers added to base-pair with ends mixture cooled; base-pairing of primers and ends of DNA strands DNA polymerases assemble new DNA strands

  37. mixture heated again; makes all DNA fragments unwind PCR (2) mixture cooled; base-pairing between primers and ends of single DNA strands DNA polymerase action again doubles number of identical DNA fragments

  38. DNA Sequencing:Reaction Mixture • Copies of DNA to be sequenced • Primer • DNA polymerase • Standard nucleotides • Modified nucleotides

  39. Reactions Proceed • Nucleotides are assembled to create complementary strands • When a modified nucleotide is included, synthesis stops • Result is millions of tagged copies of varying length

  40. T C C A T G G A C C T C C A T G G A C Recording the Sequence T C C A T G G A T C C A T G G T C C A T G T C C A T T C C A electrophoresis gel T C C • DNA is placed on gel • Fragments move off gel in size order; pass through laser beam • Color each fragment fluoresces is recorded on printout T C one of the many fragments of DNA migrating through the gel T one of the DNA fragments passing through a laser beam after moving through the gel T C C A T G G A C C A

  41. The Human Genome Project Goal - Map the entire human genome • Initially thought by many to be a waste of resources • Process accelerated when Craig Ventner used bits of cDNAs as hooks to find genes • Sequencing was completed ahead of schedule in early 2001

  42. Surprising Discoveries • Coding regions (exons) make up only 1.5% of our DNA • About half of remaining DNA is repeated segments • Around 1.4 million SNPs in the human genome (single nucleotide polymorphisms)

  43. Using Human Genes • Even with gene in hand it is difficult to manipulate it to advantage • Viruses usually used to insert genes into cultured human cells but procedure has problems • Very difficult to get modified genes to work where they should

  44. DNA Fingerprints • Unique array of DNA fragments • Inherited from parents in Mendelian fashion • Even full siblings can be distinguished from one another by this technique

  45. Tandem Repeats • Short regions of DNA that differ substantially among people • Many sites in genome where tandem repeats occur • Each person carries a unique combination of repeat numbers

  46. RFLPs • Restriction fragment-length polymorphisms • DNA from areas with tandem repeats is cut with restriction enzymes • Because of the variation in the amount of repeated DNA, the restriction fragments vary in size • Variation is detected by gel electrophoresis

  47. Biotechnology Concerns • Mutation of transgenic bacteria or viruses could yield new pathogens • Bioengineered plants and animals could alter ecological balance • Genetic screening could lead to discrimination

  48. Engineered Bacteria • Transgenic bacteria can be used to grow medically valuable proteins • Insulin, interferon, blood-clotting factors • Vaccines • Human gene is inserted into bacteria, which are then grown in huge vats

  49. Engineered Plants and Animals • Transgenic goats produce human tissue plasminogen activator (tPA) • Chinese hamster ovary cells produce blood-clotting factor VIII • Aspen plants produce less lignin and more cellulose

  50. Jefferson’s Genes • It was suspected that Jefferson had at least one child with his slave, Sally Hemings • DNA evidence showed that a modern male descendant of Sally Hemings has a Y chromosome that had been passed down to him through the generations from Thomas Jefferson

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