The Chemical Structure, Replication, and Manipulation of DNA

1. 6 The Chemical Structure, Replication, and Manipulation of DNA

2. Genome Size • Complex organisms have large genomes=genetic contents of a cell • Genomic size increases with evolutionary complexity • Size of DNA is measured in kb=kilobase pairs • Size of large genomes is measured in Mb=megabase pairs

3. DNA: Chemical Composition • Gene = unique sequence of DNA bases • Two types of nitrogen-containing bases comprise the chemical structure of DNA: - purines = adenine and guanine - pyrimidines = thymine and cytosine

5. DNA: Chemical Composition • Hydrogen bonds between purines and pyrimidines form the double-strand structure of DNA • Nucleotides = building blocks of DNA = phosphate + sugar + base • Nucleoside = sugar +base • Sugar = 5 carbon deoxyribose • Phosphodiesterbonds link sugar molecules to phosphate groups

7. DNA: Chemical Structure • DNA nucleotides = a chain of bases • Orientation of sugar-phosphate linkages = 5’to 3’ as the phosphate attached to the 5’ carbon of one sugar is linked to the 3’ carbon of the next sugar • Purine and pyrimidine bases are linked to the 1’ carbon of sugar

9. DNA: Chemical Structure • DNA consists of two polynucleotide chains which run 5’ to 3’ in opposite directions = antiparallel • DNA chains are held together by hydrogen bonds between bases • DNA bases pair by Chargaff’s rules: - Adenine (A) pairs with Thymine (T) - Guanine (G) pairs with Cytosine (C)

11. DNA: Watson-Crick Model 3-D structure of the DNA molecule: • DNA is a double helix consisting of two polynucleotide chains held together by hydrogen bonds between the purines and pyrimidines • Helix forms major and minor groove • Diameter of the helix = 20 Angstroms • Each turn of the helix = 10 bases = 34 Angstroms

13. DNA Replication Watson-Crick model of DNA replication • Hydrogen bonds between DNA bases break to allow strand separation • Each DNA strand is a template for the synthesis of a new strand • Template (parental) strand determines the sequence of bases in the new strand (daughter)= complementary base pairing rules

15. DNA Replication • Semi-conservative = each original DNA strand is a template for a new strand complementary to the original • Meselson and Stahl showed that newly synthesized DNA consists of one original strand (unlabeled) and one new strand labeled during synthesis with a heavy isotope of nitrogen

16. DNA Replication • Autoradiogram of replicating circular chromosome shows that DNA synthesis is bi-directional from a single start site = origin of replication (OR) • replication forks = region where parental strands are separating and new strands are synthesized

17. Circular DNA Replication • Movement of the replication fork is aided by topoisomerases = enzymes which unwind the DNA helix to permit strand separation • DNA topoisomerase I unwinds DNA by cutting one strand, rotating it around the second strand and then sealing the single strand break (nick)

18. Linear DNA Replication • Replication of linear DNA molecules proceeds bidirectionallyfrom multiple origins of replication which form replication loops • Replication continues to expand the replication loops until they fuse to form two separate molecules of DNA • The replicated DNAs contain the same DNA sequence

20. Rolling Circle Replication • One DNA strand is cut by a nuclease to produce a 3’-OH extended by DNA polymerase • The newly replicated strand is displaced from the template strand as DNA synthesis continues • Displaced strand is template for complementary DNA strand

22. DNA Synthesis • Addition of nucleo-tides into growing DNA chain by DNA polymerase occurs by cleavage of two phosphate groups and the attachment of the nucleoside monophosphateto the 3’-OH of adjacent deoxyribose sugar • Complementary base pairing with template specifies new strand order

24. DNA Synthesis • DNA polymerase extends a chain of nucleotides in 5’- to- 3’ direction only • Template strand is antiparallel • DNA polymerase proofreadingfunction = 3’-to-5’ exonuclease which removes mismatched bases

26. DNA Synthesis Each replication fork consists of: • Leading strand: continuous synthesis • Lagging strand: discontinuous synthesis • DNA polymerase synthesizes lagging strand in short segments = Okazaki fragments

28. DNA vs. RNA • DNA sugar = deoxyribose • RNA sugar = ribose • RNA contains the pyrimidine uracil (U) in place of thymine (T) • DNA is double-stranded • RNA is single-strand • RNA = primer to initiate DNA synthesis at origins of replication

29. Primers: Role in Replication • Primer = short RNA segment complementary to DNA at origins of replication synthesized by primase • Primer provides free 3’-OH which can be extended by DNA polymerase • Okazaki fragments are also initiated by primers eventually replaced by DNA; DNA ligase joins ends

31. DNA Replication: Proteins • Topoisomerases: nick and unwind DNA to permit strand separation • RNA primase: initiates strand synthesis by forming RNA primer • Helicase: unwinds DNA at replication fork • Single-strand binding protein: stabilize DNA at replication fork

32. DNA Replication: Proteins • DNA polymerase complex : catalyzes the incorporation of DNA nucleotides n 3’-to-5’ direction • DNA ligase: joins Okazaki fragments on lagging strand

34. Nucleic Acid Hybridization • DNA denaturation = strand separation occurs by heat to break hydrogen bonds between DNA bases • DNA renaturation = hybridization = complementary single strands pair and hydrogen bonds form • Hybridization does not occur unless DNA bases are complementary

36. Restriction Enzymes • Restriction enzymes make site specific cleavages in each DNA strand to generate “nicked” single strands with new 5’ and 3’ ends • Many enzymes cut each DNA strand at different base sites with “staggered cleavages” creating short unpaired “sticky” ends

38. Southern Blot Analysis • DNA bands on a gel can often be visualized by staining with dyes which bind DNA (ethidium bromide) • Southern blot analysis is used to detect very small amounts of DNA or to identify a single DNA band • Southern blots use labeled “probes” to identify bands by hybridizationto complementary DNA bases

40. Southern Blot Analysis Steps in Southern blot procedure: • DNA is cut into pieces by restriction enzymes • DNA fragments are separated by gel electrophoresis • DNA is transferred from gel to hybridization filter =blot procedure and denatured to produce single-strand bands of DNA

41. Southern Blot Analysis • Filter is mixed with radiolabeled single-stranded DNA probe complementary to the DNA sequence at high temperatures which permit hybridization = hydrogen bonds form between complementary base pairs • DNA bands hybridized to probe are detected by X-ray film exposure

42. Polymerase Chain Reaction • Polymerase Chain Reaction (PCR) is used to detect and amplify very small amounts of DNA • DNA sequence to be amplified is targeted using • primer oligonucleotides= short synthetic single-stranded DNA segments complementary to the DNA sequence flanking the region to be amplified

44. Polymerase Chain Reaction • Multiple short cycles of replication occurring within the region flanked by primers occurs by controlled temperature shifts which result in repetitive rounds of: - primer hybridization - DNA replication of the targeted region by primer extension - strand separation

45. Polymerase Chain Reaction • DNA polymerase used in PCR = Taq polymerase isolated from bacterial thermophiles which can withstand high temperature used in procedure • PCR accomplishes the rapid production of large amounts of target DNA which can then be identified and analyzed

46. DNA Sequence Analysis • DNA sequence analysis determines the order of bases in DNA • Dideoxy method uses DNA bases containing modified deoxyribose sugars = dideoxyribose which containH at the 3’ position of the ribose sugar rather than OH • Modified sugars cause chain termination

48. Dideoxy Method: DNA Sequencing • Each of four reactions contains a different dideoxynucleotide = A, T, G, or C in addition to the four bases • Synthesis occurs in each reaction tube until a dideoxy base is inserted which results in chain termination • Each tube contains a set of DNA pieces ending with the same base

50. Dideoxy Method: DNA Sequencing • Gel electrophoresis is used to separate the reaction products from each tube = DNAs end in A, G, T or C • DNA sequence can be read in the 5’-to-3’ direction from the bottom of gel • Each band on the gel is one base longer than the previous band • Bases are identified by gel position