Biochem March 19 Doctora Vilaran

8. Table 1: Enzymes used in molecular biology: Enzyme(s) Activity Comments Restriction endonuclease Recognizes specific nucleotide sequences; cleaves the DNA within or near the recognition sequences Reverse transcriptase (RT) RNA-dependent DNA polymerase; encoded by retrovirus Used to convert mRNA into a complementary DNA (cDNA) copy for the purpose of cloning cDNAs Rnase H Recognizes RNA-DNA duplexes; randomly cleaves the phosphodiester backbone of the RNA Used primarily to cleave the mRNA strand that is annealed to the first strand of cDNA generated by reverse transcription DNA polymerase Synthesis of DNA Used during most procedures where DNA synthesis is required; also used in vitro mutagenesis. Klenow DNA polymerase Proteolytic fragment of DNA polymerase; lacks the 5’  3’ exonuclease activity Used to incorporate radioactive nucleotides DNA fragments generated by restriction enzymes; also can be used in place of DNA polymerase DNA ligase Covalently attaches a free 5’ phosphate to a 3’ hydroxyl Used in all procedures in which two molecules of DNA need to be covalently attached

9. Table 1: Enzymes used in molecular biology: Alkaline phosphatase Removes phosphates from 5’ ends of DNA molecules Used to allow 5’ ends to be radiolabeled with the  -phosphate of ATP in the presence of polynucleotide kinase; also used to prevent self-ligation of restriction enzyme-digested plasmids and lambda vectors. Polynucleotide kinase Introduces  -phosphate of ATP to 5’ ends of DNA See above for alkaline phosphatase Dnase 1 Ramdomly hydrolyzes the phosphodiester bonds of double-stranded DNA Used in the identification of DNA regions that are bound by protein and thereby protected from Dnase 1 digestion; also used to identify transcriptionally active regions of chromatin, which are more susceptible to Dnase 1 digestion S 1 Nuclease Exonuclease that recognizes single-stranded regions of DNA Used to remove regions of single-strandedness in DNA or RNA-DNA duplexes Exonuclease III Exonuclease that removes nucleotides from the 3’ end of DNAs Used to generate deletions in DNA for sequencing or to map functional domains of DNA duplexes Terminal transferase DNA polymerase that requires only a 3’-OH; lengthens 3’ ends with any dNTP Used to introduce homopolymeric (same dNTP) “tails” onto the 3’ ends of DNA duplexes; also used to introduce radiolabeled nucleotides on the 3’ ends of DNA

10. Table 1: Enzymes used in molecular biology: T 3 , T 7 , and SP 6 RNA Polymerasesb RNA polymerase encoded by bacterial virus; recognizes specific nucleotide sequences for initiation of transcription Used to synthesize RNA in vitro Taq and Vent DNA polymerase Thermostable DNA polymerases Used in PCR Taq and Vent DNA ligases Thermostable DNA ligases Used in LCR

14. CLONING

30. POLYMERASE CHAIN REACTION

37. TWO REQUIREMENTS OF THE POLYMERASE CHAIN REACTION 1. A supply of four nucleotide bases Adenine, Guanine , Cystine and Thymine 2. Primer

39. Inherited Disorders Detected by PCR DISEASES AFFECTED GENE Adenosine deaminase deficiency Adenosine deaminase Lesch- Nyhan syndrome HGPRT Alpha1- Antitrypsin Deficiency Alpha1- Antitrypsin Fabry’s Disease Alpha-galactosidase Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Protein(CFTR) Gaucher’s Disease Glucocerebroside Sandhoff-Jatzkewitz Disease Hexosaminidase A and B Tay-sach’s Disease Hexosaminidase A

40. DISEASES AFFECTED GENE Familial Hypercholesterolemia LDL receptor Glucose-6-PhosphateDH Deficiency Glucose-6-phosphate dehydrogenase Maple Syrup urine disease Alpha keto acid decarboxylase Phenylketonuria Phenylalanine Hydroxylase Ornithine Transcarbamylase deficiency Ornithine transcarbamylase Retiniblastoma (Rb) Rb gene product Sickle – cell anemia Point mutation in Beta globin gene resulting in improper folding of protein Beta- Thalasssemia Mutations in Beta-globin gene that results in loss of synthesis of protein

41. DISEASES AFFECTED GENE Hemophilia A Factor VIII Hemophilia B Factor IX Von Willebrand Disease Von Willebrand factor

42. PCR - SSCP

43. LIGASE CHAIN REACTION

44. Sanger’s Method of DNA Sequencing

54. Since the four dyes fluoresce at different wavelengths, a laser then reads the gel to determine the identity of each band according to the wavelengths at which it fluoresces. The results are then depicted in the form of a chromatogram, which is a diagram of colored peaks that correspond to the nucleotide in that location in the sequence (Russell, 2002). Results from an automated sequence shown in the form of a chromatogram. The colors represent the four bases: yellow is C, pink is A, green is G and blue is T (Metzenberg).

55. The Fountain of Youth by Lucas Cranach

56. HYBRIDIZATION TECHNIQUES Blot Transfer procedures

57. Southern Blot A test commonly used in molecular biology and genetics, the purpose of the test being to check for a match between DNA molecules. Southern blot more formally called an DNA blot

58. Gene I Genomic DNA Restriction endonuclease Gel Electrophoresis Long DNA fragments Short DNA fragments Agarose gel DNA fragments - +

60. Autoradiography Photographic film

61. An example of a real Southern blot used to detect the presence of a gene that was transformed into a mixed cell population.

62. Northern Blot A technique in molecular biology, used mainly to separate and identify pieces of RNA. Northern blot more formally called an RNA blot.

63. Probe = cDNA Preparation of a radioactive probe: Purified mRNA + DNA primer Reverse transcriptase (DNA Polymerase) + 32 P-dNTPs DNA-RNA hybrid 1 RNase (destroy RNA) DNA-RNA hybrid Radioactive cDNA probe 2

64. Gel electrophoresis of total mRNA (agarose) Transper of RNA from gel to membrane by blotting Total RNA distribution weight Paper towel membrane Gel Wet paper towel

65. Side view: RNA is retained by the membrane Radio-active bands (hybridized probe) Radioactive RNA hybridizes only to its complementary sequence Dry and expose to X-ray film Hybridized RNA on membrane to specific DNA probe Northern blot (X-ray film) Pre mRNA Partially spliced Partially spliced mRNA

66. an example of Northern Blot

67. Western Blot A technique in molecular biology, used to separate and identify proteins. Western blot more formally called a protein immunoblot.

68. SDS Polyacrylamide Gel Electrophoresis Protein Blot on Nitrocellulose

69. Label with specific antigen Detect Antibody Reveal protein of interest

73. The DNA polymerase begins repairing the nicked DNA. Whenever a G base is read in the lower strand, a radioactive *C [light blue] base is placed in the new strand the nicked strand, as it is repaired by the DNA polymerase, is made radioactive by the inclusion of radioactive *C bases. The nicked DNA is then heated, splitting the two strands of DNA apart This creates single-stranded radioactive and non-radioactive pieces. The radioactive DNA, now called a probe [light blue], is ready for use.

75. The fit of the probe to the DNA does not have to be exact. Sequences of varying homology can stick to the DNA even if the fit is poor; the poorer the fit, the fewer the hydrogen bonds between the probe [light blue] and the denatured DNA. The ability of low-homology probes to still bind to DNA can be manipulated through varying the temperature of the hybridization reaction environment, or by varying the amount of salt in the sloshing mixture.

77. Because VNTR patterns are inherited genetically, a given person's VNTR pattern is more or less unique. The more VNTR probes used to analyze a person's VNTR pattern, the more distinctive and individualized that pattern, or DNA fingerprint, will be.

80. Restriction Map

94. new gene is injected into an adenovirus vector, which is used to introduce the modified DNA into a human cell. If the treatment is successful, the new gene will make a functional protein.

97. Steps Involved in a Human Germline Gene Therapy Protocol

99. MAKING RECOMBINANT DNA

103. DNA RECOMBINANT TECHNOLOGY SYNTHESIS OF HUMAN INSULIN

108. Thank you!!!!