Introduction To Molecular Medicine Feu (2)


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  • Introduction To Molecular Medicine Feu (2)

    1. 1. INTRODUCTION TO MOLECULAR MEDICINE <ul><li>Dolores V. Viliran, M.D. </li></ul><ul><li>Department of Biochemistry </li></ul><ul><li>and Nutrition </li></ul>
    2. 7. INTRODUCTION TO MOLECULAR MEDICINE <ul><li>DNA CLONING </li></ul><ul><li>POLYMERASE CHAIN REACTION (PCR) </li></ul><ul><li>DNA SEQUENCING </li></ul><ul><li>BLOT TECHNIQUES </li></ul><ul><li>DNA FINGERPRINTING </li></ul><ul><li>RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RLFP) </li></ul><ul><li>DNA CHIPS </li></ul><ul><li>GENE THERAPY/TRANSGENESIS </li></ul>
    3. 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
    4. 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
    5. 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
    6. 11. Characteristics of Restriction Endonucleases: <ul><li>Specific </li></ul><ul><li>They act on nucleotide sequences that are palindromic </li></ul><ul><li>Some RE make staggered symmetrical cuts away from the center of their recognition site within the DNA duplex = producing DNA with cohesive or sticky ends </li></ul><ul><li>Some RE make symmetrical cuts in the middle of their recognition site = producing fragments of DNA with blunt ends </li></ul><ul><li>Some RE cleave the DNA at a distance from the recognition sequence </li></ul>
    7. 14. CLONING
    8. 15. CLONING <ul><li>Production of large quantities of identical DNA molecules </li></ul><ul><li>A vector is required to clone either cDNAs or copies of genes. </li></ul><ul><ul><li>Classes of vectors: </li></ul></ul><ul><ul><ul><li>Plasmids </li></ul></ul></ul><ul><ul><ul><li>Lambda </li></ul></ul></ul><ul><ul><ul><li>Yeast Artificial Chromosome (YAC) vector </li></ul></ul></ul>
    9. 16. PLASMIDS <ul><li>Circular DNAs found in bacteria </li></ul><ul><li>Autonomous replication separate from the host genome </li></ul><ul><li>Contain antibiotic resistance genes that confer antibiotic resistance to bacteria </li></ul><ul><li>Limitation: Can only clone DNA less than 10,000 base pairs in length </li></ul>
    10. 17. LAMBDA <ul><li>Derived from bacteriophage (bacterial virus) </li></ul><ul><li>Bacteriophage can be classified as lysogenic or lytic </li></ul><ul><ul><li>Lysogenic- bacteriophage can integrate into the host genome </li></ul></ul><ul><ul><li>Lytic- infection followed by destruction of the infected host </li></ul></ul><ul><ul><li>Limitation: Can carry fragments of DNA up to 25,000 base pairs in length </li></ul></ul>
    11. 18. YEAST ARTIFICIAL CHROMOSOME (YAC) <ul><li>Derived from yeast </li></ul><ul><li>Use for cloning of DNA fragments up to 500,000 base pairs </li></ul>
    12. 19. STEPS IN CLONING <ul><li>Human chromosomal DNA is digested with restriction enzymes producing many DNA fragments of different sizes. Vector DNA is cleaved in the same way. </li></ul><ul><li>Vector DNA and human chromosome DNA is joined forming a recombinant DNA sealed with DNA ligase. </li></ul>
    13. 20. <ul><li>Recombinant DNA molecule is introduced into a host bacterium for replication. </li></ul><ul><li>Recombinant DNA is reproduced along with the host cell DNA. </li></ul>
    14. 21. cDNA CLONING <ul><ul><li>Production of library of cloned DNAs that represent all the mRNAs present in a particular tissue or cell. </li></ul></ul><ul><ul><li>Process begins with reverse transcription of the mRNA, followed by synthesis of the second strand of DNA and insertion of the dsDNA into a vector for cloning. </li></ul></ul><ul><ul><li>The vector is used to transform a host cell (e.g. E. coli) </li></ul></ul>
    15. 22. <ul><li>Screening of cDNA clones </li></ul><ul><ul><li>Probes: from nucleic acids or proteins </li></ul></ul><ul><ul><li>Expression cloning: with proteins, antibodies or biological assay </li></ul></ul>
    16. 23. GENOMIC CLONING <ul><li>Uses lambda-based vector systems, which are capable of carrying 15,000-25,000 bp of DNA </li></ul><ul><li>Use of a chimeric plasmid-lambda vector system termed “cosmid”: </li></ul><ul><li>Cosmid vectors contain only “cos” (cohesive) ends of the lambda genome (required for packaging the DNA into infectious virus particles </li></ul>
    17. 24. <ul><li>Digested with restriction enzymes to generate fragments in optimal size range for vector being utilized </li></ul><ul><li>Because some genes contain many more base pairs than can be inserted into a vector, overlapping clones are generated when DNA is only partially digested with restriction enzymes. </li></ul>
    18. 25. <ul><li>Partially digested DNA is size selected by a various techniques e.g. gel electrophoresis </li></ul><ul><li>Screening of genomic libraries </li></ul><ul><ul><li>Nucleic acid probes </li></ul></ul><ul><ul><li>Proteins known to bind specific sequences of DNA (e.g. transcription factors) </li></ul></ul>
    19. 26. YEAST ARTIFICIAL CHROMOSOME (YAC) CLONING <ul><li>YAC vectors allow cloning, within yeast cells, of fragments of genomic DNA that approach 500,000 base pairs. </li></ul><ul><li>Yeast vectors contain elements typical of yeast chromosomes </li></ul><ul><ul><li>Yeast centromere (CEN) </li></ul></ul><ul><ul><li>Yeast telomere (TEL) </li></ul></ul><ul><ul><li>Yeast autonomously replicating sequence (ARS) </li></ul></ul><ul><ul><li>Genes that allow the selection of yeast cells that have taken up the vector (e.g. URA3 gene invloved in uracil synthesis </li></ul></ul><ul><ul><li>Bacterial replication origin: vector can be propagated in bacterial cells </li></ul></ul><ul><ul><li>Bacterial selectable marker: e.g. gene for ampicillin resistance </li></ul></ul>
    21. 31. Polymerase Chain Reaction <ul><li>Xeroxing DNA </li></ul><ul><li>Method of amplifying a target sequence of DNA </li></ul><ul><li>Ability of DNA-copying enzyme to remain stable at high temperature </li></ul><ul><li>Can only be done in test tubes </li></ul>
    22. 32. Polymerase Chain Reaction <ul><li>Was first described by Dr. Karry Mullis , and it was this invention that he received a Nobel Prize in Medicine and Physiology in 1993. </li></ul><ul><li>In vitro enzymatic amplification of specific DNA sequence which involves three steps which are repeated a number of times(cycle) </li></ul>
    23. 33. Three parts of the Polymerase Chain Reaction <ul><li>1. DNA Denaturation </li></ul><ul><ul><li>The double-stranded template DNA ( usually genomic DNA) is dissociated into single strands by heating the sample at 92 – 94 o C (process of separation) </li></ul></ul>
    24. 34. <ul><li>2. PRIMER Annealing </li></ul><ul><li>By lowering the temperature to 40 – 60 o C, two oligonucleotide primers (typically 18-22 bases in length) can anneal to regions on the single DNA strand that flank the target DNA sequence to be amplified. </li></ul>
    25. 35. <ul><li>PRIMER Extensions </li></ul><ul><li>The 3 ’ end of the oligonucleotide primers are extended toward each other with newly synthesized DNA . This new DNA is complementary to target DNA sequences. To reduce non specific annealing of primers to DNA, this step is usually performed at an elevated temperature (e.g. 72 o C using a thermostable DNA polymerase – typically Taq DNA polymerase from Thermus aquaticus) </li></ul>
    26. 37. TWO REQUIREMENTS OF THE POLYMERASE CHAIN REACTION 1. A supply of four nucleotide bases Adenine, Guanine , Cystine and Thymine 2. Primer
    27. 38. PCR TEST <ul><li>- investigate a potentially defective DNA sequence </li></ul><ul><li>- diagnosis of diseases in which a specific mutational site is in question </li></ul><ul><li>- primers spanning the site are used </li></ul><ul><li>-the amplified DNA can de sequenced or otherwise compared with the wild type </li></ul>
    28. 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
    29. 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
    30. 41. DISEASES AFFECTED GENE Hemophilia A Factor VIII Hemophilia B Factor IX Von Willebrand Disease Von Willebrand factor
    31. 42. PCR - SSCP
    33. 44. Sanger’s Method of DNA Sequencing
    34. 46. Chain Termination Method <ul><li>Based on DNA polymerase reaction </li></ul><ul><li>Run four separate reactions </li></ul><ul><li>Each reaction mixture contains dATP, dGTP, dCTP and dTTP, one of which is P-32-labelled </li></ul><ul><li>Each reaction also contains a small amount of one dideoxynucleotide : either dd ATP, dd GTP, dd CTP or dd TTP </li></ul>
    35. 47. Chain Termination Method <ul><li>Most of the time, the polymerase uses normal nucleotides and DNA molecules grow normally </li></ul><ul><li>Occasionally, the polymerase uses a dideoxynucleotide, which adds to the chain and then prevents further growth in that molecule </li></ul><ul><li>Random insertion of dd-nucleotides leaves (optimally) at least a few chains terminated at every occurrence of a given nucleotide </li></ul>
    36. 48. Chain Termination Method <ul><li>Run each reaction mixture on electrophoresis gel </li></ul><ul><li>Short fragments go to bottom, long fragments on top </li></ul><ul><li>Read the &quot;sequence&quot; from bottom of gel to top </li></ul><ul><li>Convert this &quot;sequence&quot; to the complementary sequence </li></ul><ul><li>Now read from the other end and you have the sequence you wanted - read 5' to 3' </li></ul>
    37. 50. Chemical Cleavage Method <ul><li>Not used as frequently as Sanger's </li></ul><ul><li>Start with ssDNA labelled with P-32 at one end </li></ul><ul><li>Strand is cleaved by chemical reagents </li></ul><ul><li>Assumption is that strands of all possible lengths, each cleaved at just one of the occurrences of a given base, will be produced. </li></ul><ul><li>Fragments are electrophoresed and sequence is read </li></ul>
    38. 51. Chemical Cleavage Method <ul><li>Four reactions are used </li></ul><ul><li>G -specific cleavage with dimethyl sulfate , followed by strand scission with piperidine </li></ul><ul><li>G/A cleavage: depurination with mild acid , followed by piperidine </li></ul><ul><li>C/T cleavage: ring hydrolysis by hydrazine , followed by piperidine </li></ul><ul><li>C cleavage: same method ( hydrazine and piperidine ), but high salt protects T residues </li></ul>
    39. 52. Chemical Cleavage Method <ul><li>Reading the gels... </li></ul><ul><li>It depends on which end of the ssDNA was radioactively labelled! </li></ul><ul><li>If the 5'-end was labelled, read the sequence from bottom of gel to top (5' to 3') </li></ul><ul><li>If the 3'-end was labelled, read the sequence from top of gel to bottom (5' to 3') </li></ul><ul><li>Note that the nucleotide closest to the P-32 will be missed in this procedure </li></ul>
    40. 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).
    41. 55. The Fountain of Youth by Lucas Cranach
    42. 56. HYBRIDIZATION TECHNIQUES Blot Transfer procedures
    43. 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
    44. 58. Gene I Genomic DNA Restriction endonuclease Gel Electrophoresis Long DNA fragments Short DNA fragments Agarose gel DNA fragments - +
    45. 59. DNA fragments Agarose gel - + <ul><li>Denature in alkali </li></ul><ul><li>Blot-transfer bake </li></ul>ssDNA fragments nitrocellulose Radioactive RNA or denatured DNA containing sequences complementary to gene I (radioactive probe) <ul><li>Hybridize nitrocellulose with radioactive probe </li></ul><ul><li>wash </li></ul>
    46. 60. Autoradiography Photographic film
    47. 61. An example of a real Southern blot used to detect the presence of a gene that was transformed into a mixed cell population.
    48. 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.
    49. 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
    50. 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
    51. 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
    52. 66. an example of Northern Blot
    53. 67. Western Blot A technique in molecular biology, used to separate and identify proteins. Western blot more formally called a protein immunoblot.
    54. 68. SDS Polyacrylamide Gel Electrophoresis Protein Blot on Nitrocellulose
    55. 69. Label with specific antigen Detect Antibody Reveal protein of interest
    56. 70. <ul><li>DNA fingerprinting is a system identifying organisms, disease and paternity based on DNA analysis </li></ul><ul><li>use to solve crimes by identifying the victim, criminal and other participants in a criminal act. </li></ul>
    57. 71. Procedure: <ul><ul><li>Performing a Southern Blot </li></ul></ul><ul><ul><li>Making a Radioactive Probe </li></ul></ul><ul><ul><li>Creating a Hybridization Reaction </li></ul></ul><ul><ul><li>VNTRs </li></ul></ul>
    58. 72. <ul><ul><ul><li>Performing a Southern Blot </li></ul></ul></ul><ul><ul><li>Making a Radioactive Probe </li></ul></ul>Introduce horizontal breaks along a strand, , add individual nucleotides to the nicked DNA, one of which, *C [light blue], is radioactive. Add the DNA polymerase DNA polymerase will become immediately attracted to the nicks in the DNA and attempt to repair the DNA, starting from the 5' end and moving toward the 3' end
    59. 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.
    60. 74. <ul><li>Creating a Hybridization Reaction </li></ul>Hybridization is the coming together, or binding, of two genetic sequences. The binding occurs because of the hydrogen bonds [pink] between base pairs. DNA must first be denatured, usually by using heat or chemicals . The denatured DNA is put into a plastic bag along with the probe and some saline liquid; the bag is then shaken to allow sloshing. If the probe finds a fit, it will bind to the DNA
    61. 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.
    62. 76. <ul><ul><li>VNTRs </li></ul></ul><ul><li>Every strand of DNA has exons and introns </li></ul><ul><li>introns contain repeated sequences of base pairs called Variable Number Tandem Repeats (VNTRs) . (twenty to one hundred base pairs) </li></ul><ul><li>Every human being has some VNTRs </li></ul><ul><li>To determine if a person has a particular VNTR, a Southern Blot is performed, and then the Southern Blot is probed, through a hybridization reaction, with a radioactive version of the VNTR in question </li></ul><ul><li>The pattern which results from this process is what is often referred to as a DNA fingerprint </li></ul><ul><li>A given person's VNTRs come from the genetic information donated by his or her parents; he or she could have VNTRs inherited from his or her mother or father, or a combination, but never a VNTR either of his or her parents do not have </li></ul>
    63. 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.
    64. 78. Applications of DNA fingerprinting <ul><li>Paternity and Maternity </li></ul><ul><li>Criminal Identification and Forensics </li></ul><ul><li>Personal Identification </li></ul>
    65. 80. Restriction Map
    66. 81. Restriction fragment length polymorphism (RFLP) <ul><li>Restriction fragment length polymorphism is the identification of specific restriction enzymes that reveal a pattern difference between the DNA cut DNA at specific 4-6 bp recognition sites </li></ul><ul><li>Sample DNA is cut with one or more RE’s and resulting fragments are separated according to molecular size using gel electrophoresis </li></ul><ul><li>Differences result from base substitutions, additions, deletions or sequence rearrangements within RE recognition sequences </li></ul><ul><li>Presence and absence of fragments resulting from changes in recognition sites are used identifying species or populations. </li></ul>
    67. 82. Applications <ul><li>This can be used to genetically tell individuals apart. </li></ul><ul><li>It can show the genetic relationship between individuals, because children inherit genetic elements from their parents. </li></ul><ul><li>It is used to determine the relationships among species. </li></ul>
    68. 83. Restriction enzymes cut DNA at precise points producing <ul><li>A collection of DNA fragments of precisely defined length. </li></ul><ul><li>These can be separated by electrophoresis with smaller fragments migrating farther than the larger fragments. </li></ul><ul><li>One or more fragments can be visualized with a “probe” – molecule of single stranded DNA that is </li></ul><ul><ul><ul><li>Complementary to a run of nucleotides in one or more of the restriction fragments and is </li></ul></ul></ul><ul><ul><ul><li>Radioactive (or fluorescent) </li></ul></ul></ul>
    69. 84. Cases where RFLP is use <ul><li>Sickle-cell disease </li></ul><ul><li>The only difference between the </li></ul><ul><li>two genes is the substitution of a T </li></ul><ul><li>or an A in the middle position of </li></ul><ul><li>codon 6 </li></ul><ul><li>A change of a single nucleotide </li></ul><ul><li>produced the RFLP </li></ul><ul><li>This is a very common cause of </li></ul><ul><li>RFLPs also called as a single </li></ul><ul><li>Nucleotide polymorphisms or </li></ul><ul><li>SNPs </li></ul><ul><li>Screening for Sickle-cell anemia </li></ul>
    70. 85. DNA Microchips <ul><li>a mutation - in a particular gene's DNA often results in a certain disease </li></ul><ul><li>The DNA microchip is a revolutionary new tool used to identify mutations in genes </li></ul><ul><li>The chip, which consists of a small glass plate encased in plastic, is manufactured somewhat like a computer microchip </li></ul><ul><li>On the surface, each chip contains thousands of short, synthetic, single-stranded DNA sequences, which together, add up to the normal gene in question. </li></ul>
    71. 86. Use of DNA Microchips <ul><li>Research tool </li></ul><ul><li>As we gain more insight into the mutations that underly various diseases </li></ul><ul><li>DNA chips can help assess individual risks for developing different cancers as well as heart disease, diabetes and other diseases. </li></ul>
    72. 87. Procedure <ul><li>determine whether an individual possesses a mutation for certain genes, a scientist first obtains a sample of DNA from the patient's blood as well as a control sample </li></ul><ul><li>denatures the DNA in the samples - a process that separates the two complementary strands of DNA into single-stranded molecules. </li></ul><ul><li>cut the long strands of DNA into smaller, more manageable fragments and then to label each fragment by attaching a fluorescent dye. The individual's DNA is labeled with green dye and the control - or normal - DNA is labeled with red dye. Both sets of labeled DNA are then inserted into the chip and allowed to hybridize </li></ul><ul><li>If the individual does not have a mutation for the gene, both the red and green samples will bind to the sequences on the chip. </li></ul><ul><li>If the individual does possess a mutation, the individual's DNA will not bind properly in the region where the mutation is located. The scientist can then examine this area more closely to confirm that a mutation is present. </li></ul>
    73. 88. DNA CHIPS - Microarray technology <ul><li>This technology promises to monitor the whole genome on a single chip so that researchers can have a better picture of the interactions among thousands of genes simultaneously. </li></ul><ul><li>Principle: </li></ul><ul><li>Base-pairing (i.e., A-T and G-C for DNA; A-U and G-C for RNA) or hybridization is the underlining principle of DNA microarray. </li></ul><ul><li>The sample spot sizes in microarray are typically less than 200 microns in diameter and these arrays usually contains thousands of spots. </li></ul>
    74. 89. Application <ul><li>Identification of sequence (gene/ gene mutation) </li></ul><ul><li>determination of expression level (abundance) of genes. </li></ul><ul><li>The microarray (DNA chip) technology is having a significant impact on genomics study Many fields, including drug discovery and toxicological research, will certainly benefit from the use of DNA microarray technology. </li></ul><ul><li>Disease diagnosis </li></ul><ul><li>Drug discovery: Pharmacogenomics </li></ul>
    75. 90. <ul><li>Gene therapy is a technique for correcting defective genes responsible for disease development. </li></ul><ul><li>Gene therapy is designed to introduce genetic material into cells to compensate for abnormal genes or to make a beneficial protein. If a mutated gene causes a necessary protein to be faulty or missing, gene therapy may be able to introduce a normal copy of the gene to restore the function of the protein. </li></ul>
    76. 91. Several approaches for correcting faulty genes <ul><li>A normal gene may be inserted into a nonspecific location within the genome to replace a nonfunctional gene. This approach is most common. </li></ul><ul><li>An abnormal gene could be swapped for a normal gene through homologous recombination. </li></ul><ul><li>The abnormal gene could be repaired through selective reverse mutation, which returns the gene to its normal function. </li></ul><ul><li>The regulation (the degree to which a gene is turned on or off) of a particular gene could be altered. </li></ul>
    77. 92. <ul><li>The most common techniques utilized in gene therapy studies is the introduction of the corrected gene into bone marrow cells, skin fibroblasts or hepatocytes. The vectors most commonly utilized are derived from retroviruses and utilize only the transcriptional promoter regions of these viruses (the LTRs) to drive expression of the gene of interest. The advantage of retroviral-based vector systems is that expression occurs in most cell types. </li></ul><ul><li>A number of human inherited disorders have been corrected in cultured cells and several diseases (e.g. malignant melanoma and severe combined immunodeficiency disease, SCID) are currently being treated by gene therapy techniques indicating that gene therapy is likely to be a powerful therapeutic technique against a host of diseases in coming years . </li></ul>
    78. 93. <ul><li>A gene that is inserted directly into a cell usually does not function. Instead, a carrier called a vector is genetically engineered to deliver the gene. Certain viruses are often used as vectors because they can deliver the new gene by infecting the cell. The viruses are modified so they can't cause disease when used in people. Some types of virus, such as retroviruses, integrate their genetic material (including the new gene) into a chromosome in the human cell. Other viruses, such as adenoviruses, introduce their DNA into the nucleus of the cell, but the DNA is not integrated into a chromosome. </li></ul><ul><li>The vector can be injected or given intravenously (by IV) directly into a specific tissue in the body, where it is taken up by individual cells. Alternately, a sample of the patient's cells can be removed and exposed to the vector in a laboratory setting. The cells </li></ul><ul><li>containing the vector are then returned to the patient. If the treatment is successful, the new gene delivered by the vector will make a functioning protein. </li></ul>
    79. 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.
    80. 95. Some of the different types of viruses used as gene therapy vectors: <ul><li>Retroviruses </li></ul><ul><li>Adenoviruses </li></ul><ul><li>Adeno-associated viruses </li></ul><ul><li>Herpes simplex viruses </li></ul>
    81. 96. <ul><li>Current gene therapy research has focused on treating individuals by targeting the therapy to body cells such as bone marrow or blood cells. This type of gene therapy cannot be passed on to a person’s children. Gene therapy could be targeted to egg and sperm cells (germ cells), however, which would allow the inserted gene to be passed on to future generations. This approach is known as germline gene therapy .(TRANSGENESIS) </li></ul>
    82. 97. Steps Involved in a Human Germline Gene Therapy Protocol
    83. 98. <ul><li>Isolation of totipotent embryonic cells at an undifferentiated stage </li></ul><ul><li>Determination of the genetic state of the embryo . </li></ul><ul><li>Expansion of embryonic stem cells in culture . </li></ul><ul><li>Transfer of genetic material into embryonic cells </li></ul><ul><li>Selection of cells which have stably taken up the transfected gene </li></ul><ul><li>Targeted gene replacement </li></ul><ul><li>Marker removal . </li></ul><ul><li>Confirming genomic integrity </li></ul><ul><li>Nuclear transfer </li></ul><ul><li>Reimplantation into the mother </li></ul>
    85. 100. Uses <ul><li>Medicine </li></ul><ul><li> - Genetic engineering is able to treat many illnesses and conditions with the human body which were previously much more harmful. Many medicines and treatments are available only because of this technology </li></ul>
    86. 101. <ul><li>Agriculture </li></ul><ul><li>-Much of the food we eat are in some way connected to genetic engineering. In fact, about 60 percent of our food has some sort of biotechnology in it. By taking traits from one organism and putting it into a food, the food can be altered in many ways, like having it last longer, taste better, and grow faster and larger. It can also be designed to be more immune to certain diseases. </li></ul>
    87. 102. <ul><li>Industry </li></ul><ul><li>- The ability of bacteria to produce chemicals can be used in other areas as well such as in the cheese industry. Also, genetically related advances and business is booming. </li></ul><ul><li>Terrorism </li></ul><ul><li>Former Soviet Union used traits in many organisms to make biological weapons and viruses never before heard of. One scenario is that by using recombinant DNA methods, they might have taken the lethality of Ebola, combined it with the worst part of anthrax, and to top it off, made it extremely contagious by including parts of the smallpox virus. </li></ul>
    89. 104. What are Biotechnology Drugs? <ul><li>Drugs produced using living organisms such as yeast , bacteria , or mammalian cells. </li></ul><ul><li>Examples: </li></ul><ul><ul><li>Cytokines </li></ul></ul><ul><ul><li>Human Growth Hormone ( hGH) </li></ul></ul><ul><ul><li>Insulin </li></ul></ul><ul><ul><li>Interferon </li></ul></ul><ul><ul><li>Factor </li></ul></ul><ul><ul><li>Erythropoietin </li></ul></ul><ul><ul><li>Monoclonal Antibodies </li></ul></ul>
    90. 105. Approved Biotechnology Drugs 1999 <ul><li>1. Activase (Alteplase recombinant): treatment of acute myocardial infarction , acute massive pulmonary embolism, acute ischemic stroke within the first hours of symptom onset. </li></ul><ul><li>2. Alphanate ( human antihemophilic factor): treatment of hemophilia or acquired Factor VII deficiency. </li></ul>
    91. 106. <ul><li>3. Humalog ( recombinant insulin) : treatment of diabetes. </li></ul><ul><li>4. Geref: treatment of growth hormone deficiency in children with growth failure. </li></ul><ul><li>5. GenoTropin: treatment of growth hormone deficiency in children; growth deficiency in adults. </li></ul>
    92. 107. <ul><li>6.Vistide(cidofovir injection)/ Vitravene (fomivirsen sodium, injectable): treatment of (CMV) retinitis in AIDS. </li></ul><ul><li>7.Zenapax (Daclizumab): humanized monoclonal antibody for prevention of kidney transplant rejection. </li></ul><ul><li>(The medicines and vaccines included in this list are produced and/or developed by companies involved in recombinant DNA research or other biotechnology applications.). </li></ul>
    93. 108. Thank you!!!!