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Medical biotechnology
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Health biotechnology

  1. 1. HEALTH BIOTECHNOLOGY Rizwan Abbas baho
  2. 2. Course Contents: Introduction to Health biotechnology Social acceptance of medical biotechnology The molecular basis of disease, Molecular and genetic markers Detection of infectious agents Active and passive immunization vaccines ,Organ transplantation, Applications of transgenic animals Drug delivery systems, Blood transfusion, Grafting techniques, Pharmacogenetics, Strategies of gene therapy, gene delivery vehicles, Biopharmaceuticals from plants Uses of stem cell technology
  3. 3. Reference Books “Medical Biotechnology” by Judit Pongracz, Mary Keen “(2009). Elsevier Health Sciences. “Biotechnology and Your Health: Pharmaceutical Applications” by Bernice Zeldin Schacter, Bernice Schacter (2005) Chelsea House Publishers, “Health and Pharmaceutical Biotechnology” by D.M. Chetan, K.P. Dinesh, D.M. Chetan (2006) Firewall Media.
  4. 4.  Introduction to health biotechnology  Applications  Drug production  Pharmacogenomics  Gene therapy  Genetic testing OUTLINE (Lecture-I)
  5. 5. What Is Biotechnology?  Scientific processes to get new organisms or new products from organisms.  It is the use of living organisms or processes to develop products useful for mankind.
  6. 6.  Has been existing since centuries  Begin with the first action of human on life for his welfare  Term coined by a Hungarian engineer Karl Ereky  Modern biotechnology started in California in 1970’s History
  7. 7. Origins of Biotechnology  Although it seems like a new thing, biotechnology has actually been around for a while:  Domesticated plants and animals are the result of selective breeding  Using yeast to make bread rise  Using bacteria or yeast to ferment grapes into wine
  8. 8. Any technique that uses living organisms or substances from those organisms to make or modify a product, to improve plants or animals or to develop microorganisms for specific uses Definition
  9. 9. Green biotechnology (agricultural) Red biotechnology (medical) Blue biotechnology (aquatic) White biotechnology (industrial) Applications
  10. 10.  The use of biological methods to optimize industrial processes  Applied by manufacturers of laundry detergents  Includes research for new enzymes (proteins that remove oily and protein-based stains)  Enzymes that work under extreme conditions (wash temperatures of 20°C or 90°C)  This often entails modifying the enzymes of microorganisms for these processes White biotechnology
  11. 11.  Use of biotechnological techniques in agriculture  Vitamin A deficiency is a serious problem and can cause blindness at a young age if left untreated  Golden rice was genetically modified to produce beta-carotene (a precursor of vitamin A that the body converts to vitamin A). A diet including golden rice can thus help to raise vitamin A levels Green biotechnology:
  12. 12.  Also called red biotechnology  It includes: o Production of medicines and pharmaceutical products for treating or diagnosing disorders o Designing of organisms to manufacture antibiotics and vaccines o Engineering of genetic defects through genomic manipulation o Use in forensics through DNA profiling Biotechnology and medicine:
  13. 13.  Production of human insulin from non- human sources.  Production of hormones like Interferons, Cytokinins, Steroids and human growth hormones.  Gene therapy for prevention and control of diseases like hemophilia cystic fibrosis  Development of vaccines and antibodies for rabies, HIV, etc. Examples…
  14. 14.  Drug production  Pharmacogenomics  Gene therapy  Genetic testing Health Biotechnology
  15. 15.  It is the process in which pharmaceutical products are produced through application of biotechnological techniques  Medicines are produced for: • Diagnosis • Cure treatments • Prevention of diseases Drug production
  16. 16.  Producing medicines through:  Isolating enzymes  Genetically engineering enzymes Drug production
  17. 17.  Recently, plants are being genetically modified to produce pharmaceutical products instead of their natural compounds  For Example: A drug Elelyso for treating Gaucher is being produced by genetically engineering carrots Drug production
  18. 18.  INSULIN: Human insulin is being produced using genetic engineering technique known as humulin and it is used for the treatment of diabetes that is low sugar level in the blood….. Drug production
  19. 19.  INTERFERON: o Interferon interfere in transmission of viral genome from one cell to another and it also inhibits the cell division of abnormal cells. o Interferon produced using the recombinant DNA technology is used to treat cancer patients. o Interferon improved the quality of life of cancer patients….. Drug production
  20. 20.  HUMAN GROWTH HORMONE: Since dwarfism is caused by growth hormone deficiency so it can be diagnose by HGH testing. So HGH is used for the treatment of dwarfism due to hypo pituitary activity. Drug production
  21. 21. Pharma = Drug or Medicine Genomics = The study of genes Studying response of genetic make up of an individual to a drug or pharmaceutical products Pharmacogenomics
  22. 22.  “One-size-fits-all drugs” only work for about 60 percent of the population at best. And the other 40 percent of the population increase their risks of adverse drug reaction because their genes do not do what is intended of them. Use of Pharmacogenomics:
  23. 23.  Helps in the development of tailor made medicines  Ensures more appropriate methods of determining drug dosages  Improve process of drug discovery and approval  Obtaining of better and safer vaccination  Decrease in the overall cost of Health Care  Advanced Screening for Disease Impotance Of Pharmacogenomics
  24. 24. Opinion:  This sort of card would initially (~2025) include mostly information related to drug metabolizing enzymes.  Around ~2050 it might include an entire individual genome Pharmacogenomics SMART CARD (Confidential)
  25. 25. Some barriers faced are: Complexity of finding gene variation that affect drug response Limited drug alternatives Disincentives for drug companies to make multiple pharmacogenomic products Educating healthcare providers Pharmacogenomics
  26. 26.  The process in which a faulty gene is removed or replaced with its healthy copy to restore the normal function of that gene Gene therapy
  27. 27.  Replacing a mutated gene that causes disease with a healthy copy of the gene  Inactivating or “knocking out” a mutated gene that is functioning improperly  Introducing the new gene that help fight a disease Gene therapy
  28. 28.  Some common ways are:  Using fat droplets in nose sprays  Using cold viruses that are modified to carry alleles ,go into the cell and affect them  The direct injection of DNA(might include electroporation or biolistic method) Gene therapy
  29. 29. The process of gene therapy is of two types:  Stem cell gene therapy: In this gene therapy is applied on a fully developed organism and the effects of gene therapy lasts only to the operated organism  Germ line gene therapy: In this process gene therapy is done on a fertilized egg or an early embryo and the altered genome is followed in next generations. Gene therapy
  30. 30. Gene therapy
  31. 31. 4) Tissue Engineering  A form of regenerative medicine, tissue engineering is the creation of human tissue outside the body for later replacement.  Usually occurs on a tissue scaffold, but can be grown on/in other organisms as shown on the right.
  32. 32. 4) Tissue Engineering  Tissue engineers have created artificial skin, cartilage and bone marrow.  Current projects being undertaken include creating an artificial liver, pancreas and bladder.  Again, we are far from replacing a whole organ, but just looking for “refurbishing” our slightly used ones at the moment.
  33. 33.  The examination of a patient’s DNA molecule to determine his/her DNA sequence for mutated genes  The genome of an individual is scaned for this purpose by a scientist Genetic testing
  34. 34.  Forensic/identity testing  Determining sex  Conformational diagnosis of symptomatic individuals  Newborn screening  Prenatal diagnostic screening Genetic testing
  35. 35.  Better drugs can be obtained by the knowledge of genetics  Genetic testing can be used to detect the mutations regarding genetic disorders like cystic fibrosis, sickle cell anaemia, hutington diseases, etc.  Tests are also being developed to detect various cancers Genetic testing
  36. 36. Mutation Detection Dr. Sajjad Ahmad
  37. 37. Mutations Detection Detection of mutations has central role in various areas of genetic diagnosis  Preimplantation genetic diagnosis (PGD),  Prenatal diagnosis (PND)  Presymptomatic testing  Confirmational diagnosis  Forensic/identity testing. Two groups of tests, molecular and cytogenetic, are used in genetic syndromes.
  38. 38. Single Base Pair Mutations Direct sequencing, DNA hybridization and/or restriction enzyme digestion methods are used for detection of single pair mutations. However, there are two approaches for genetic diagnosis;  Indirect approach depends on the results from a genetic linkage analysis using DNA markers such as STR(short tandem repeat) or VNTR (variable number tandem repeat) markers flanking or within the gene  Direct approach for diagnosis essentially depends on the detection of the genetic variations responsible for the disease
  39. 39. Cytogenetics and Molecular Diagnostics  Karyotyping  Fluorescence in situ hybridization (FISH)  Comparative genomic hybridization (CGH)  Molecular Diagnostics (Known & Unknown Mutations)  Next Generation Sequencing (NGS)
  40. 40. Karyotyping  Karyotyping is the process of pairing and ordering all the chromosomes of an organism, thus providing a genome-wide image of an individuals chromosomes  Karyotypes are prepared from mitotic cells which are frozen in metaphase.  Characteristic structural features for each chromosome are revealed.  Can reveal changes in chromosome numbers linked to conditions such as Down’s syndrome.  Careful analysis can show more subtle changes as chromosomal deletions, duplications, translocations or inversions.  There is an increasing use of karyotyping for diagnosis of specific birth defects and genetic disorders.
  41. 41. Karyotyping Applications Chromosome studies are advised in the following situations:  Suspected chromosome abnormality  Sexual disorders  Multiple congenital anomalies/ developmental retardation  undiagnosed learning disabilities  Infertility or multiple miscarriage  Stillbirth and malignancies
  42. 42. Preparation of visual karyotype  Traditionally, the microscopic study of chromosomes is performed on compacted chromosomes at a magnification of 1000 at metaphase.  Cells are arrested at metaphase stage with a microtubule polymerization inhibitor such as colchicine  These cells are spread on a glass slide and stained with Giemsa stain (G banding).  Chromosomes are studied by making a photograph or digital imaging and subsequent assembling of chromosomes
  43. 43. Process of Karyotyping
  44. 44. Human Karyotype Human chromosomes are categorized based on position of centromere;  Metacentric; the centromere at center (chromosomes 1, 3, 16, 19 and 20),  Acrocentric; the centromere near one end (chromosomes 13, 14, 15, 21, 22 and Y are)  other chromosomes are submetacentric  The convenient methods of chromosome banding are G-(Giemsa), R-(reverse),C-(centromere) and Q- (quinacrine) banding
  45. 45. Fluorescence in situ hybridization (FISH)
  46. 46. Fluorescence in situ hybridization (FISH):  FISH is applied to provide specific localization of genes on chromosomes.  Rapid diagnosis of trisomies and microdeletions is acquired using specific probes.  Usually a denatured probe is added to a metaphase chromosome spread and incubated overnight to allow sequence-specific hybridization.  After washing off the unbound probe, the bound probe is visualized by its fluorescence under UV light; thus, the site of the gene of interest is observed as in situ
  47. 47. Comparative genomic hybridization (CGH)
  48. 48. Comparative genomic hybridization (CGH)  CGH, a special FISH technique (dual probes), is applied for detecting all genomic imbalances.  The basics of technique is comparison of total genomic DNA of the given sample DNA (e.g. tumor DNA) with total genomic DNA of normal cells.  Typically, an identical amount of both tumor and normal DNAs is labeled with two different fluorescent dyes; the mixture is added and hybridized to a normal lymphocyte metaphase slide.  A fluorescent microscope equipped with a camera and an image analysis system are used for evaluation  Copy number of genetic material (gains and losses) is calculated by evaluation software.
  49. 49.  CGH is used to determine copy number alterations of genome in cancer and those cells whose karyotype is hard or impossible to prepare or analyze.  In array-CGH, metaphase slide is replaced by specific DNA sequences, spotted in arrays on glass slides, so its resolution is increased. Comparative genomic hybridization (CGH)
  50. 50. Molecular Diagnostics
  51. 51. Molecular Diagnostics  Molecular methods for identification of the disease- causing mutations could be classified as methods for known and methods for unknown mutations. Several criteria, have to be met for choosing a suitable method; for example  type of nucleic acid (DNA or RNA)  kind of specimen (blood, tissues, etc.)  the number of mutations  reliability of the method
  52. 52. Detection of Known Mutations Many different approaches have been used for identifying known mutations  Polymerase chain reaction (PCR) and its versions  DNA microarray  DNA Sequencing  Multiplex ligation-dependent probe amplification (MLPA)
  53. 53. Detection of Unknown Mutations  Single Strand Conformational Polymorphism (SSCP)  Denaturing Gradient Gel Electrophoresis (DGGE)  Restriction fragment length polymorphism (RFLP)
  54. 54. 1. Polymerase chain reaction  In 1980s, Dr Mullis introduced a method for amplifying DNA fragment to a large number of fragments by polymerase chain reaction (PCR)  Essential components of PCR are template DNA, primers , thermostable DNA polymerase enzyme (e.g. Taq), divalent cations (usually Mg2+), deoxynucleoside triphosphates (dNTPs) and buffer solution  PCR, consisting of 25-40 repeated cycles, has three discrete steps of temperature changes
  55. 55. Steps of PCR  Initial denaturation step includes heating the reaction to a temperature of 92–96°C for 1–9 minutes.  1) Denaturation step includes heating the reaction to 92–98°C for 20– 30 seconds. The hydrogen bonds between complementary bases are disrupted and DNA molecules are denatured, yielding single-stranded DNA molecules (DNA melting).  2) Annealing step is performed by decreasing temperature to 50– 65°C for 25–40 seconds; so the primers are annealed to their targets on single stranded DNAs by hydrogen bonds and a polymerase can bind to the primer-template hybrid and begin DNA polymerization in next step.  3) Extension step includes polymerization of the bases to the primers; a thermostable such as Taq polymerase extends a new strand complementary to the DNA template strand by adding matched dNTPs in 5' to 3' direction at a temperature of 72°C.  A series of 25-40 repeated cycles of denaturation, annealing of primers and extension is performed to amplify the template fragment.  Subsequently, a final elongation is sometimes done at 70–74°C for 5– 15 minutes after the last PCR cycle to ensure full extension of any remaining single-stranded DNA
  56. 56. Types and Applications of PCR 1) Reverse transcriptase PCR (RT-PCR) 2) Multiplex PCR 3) Nested PCR 4) Amplification refractory mutation system (ARMS) PCR: 5) Real time PCR
  57. 57. 1. Reverse transcriptase PCR (RT-PCR) In this version, a strand of RNA molecule is transcribed reversely into its complementary DNA (cDNA) using the reverse transcriptase enzyme. This cDNA is then amplified by PCR. RT-PCR is applied to study the mutations at RNA level.
  58. 58. 2) Multiplex PCR: In this technique, multiple selected target regions in a sample are amplified simultaneously using different pairs of primers. 3) Nested PCR: It includes two successive PCRs; the product of the first PCR reaction is used as a template for the second PCR. This type of PCR is employed to amplify templates in low copy numbers in specimens. It has the benefits of increased sensitivity and specificity.
  59. 59. 4) Amplification refractory mutation system (ARMS) PCR: Allele-specific amplification (AS-PCR) or ARMS-PCR is a general technique for the detection of any point mutation or small deletion The genotype (normal, heterozygous and homozygous states) of a sample could be determined using two complementary reactions: one containing a specific primer for the amplification of normal DNA sequence at a given locus and the other one containing a mutants pecific primer for amplification of mutant DNA. ARMS-PCR has been used to check the most common mutation in GJB2 gene, 35delG mutation among deaf children. 5) Real time PCR: In this technique, the amplified DNA is detected as the PCR progresses. It is commonly used in gene expression studies and quantification of initial copy number of the target
  60. 60. DNA microarray  DNA “chips” or microarrays have been used as a possible testing for multiple mutations  Single DNA strands including sequences of different targets are fixed to a solid support in an array format.  On the other hand, the sample DNA or cDNA labeled with fluorescent dyes is hybridized to the chip  Then using a laser system, the presence of fluorescence is checked; the sequences and their quantities in the sample are determined
  61. 61. DNA Sequencing The main aim of DNA sequencing is to determine the sequence of small regions of interest (~1 kilobase) using a PCR product as a template.  Dideoxynucleotide sequencing or Sanger sequencing represents the most widely used technique for sequencing DNA  In this method, double stranded DNA is denatured into single stranded DNA with NaOH  A Sanger reaction consists of a single strand DNA, primer, a mixture of a particular ddNTP with normal dNTPs (e.g. ddATP with dATP, dCTP, dGTP, and dTTP).  A fluorescent dye molecule is covalently attached to the dideoxynucleotide. ddNTPs cannot form a phosphodiester bond with the next deoxynucleotide so that they terminate DNA chain elongation.  This step is done in four separate reactions using a different ddNTP for each reaction  DNA sequencing could be used to check all small known and unknown DNA variations.
  62. 62. Multiplex ligation-dependent probe amplification (MLPA)  MLPA is commonly applied to screen deletions and duplications of up to 50 different genomic DNA or RNA sequences.  Altogether gene deletions and duplications account up to 10%, and in many disorders up to 30% of disease-causing mutations  The probe set is hybridized to genomic DNA in solution  Each probe consists of two halves; one half is composed of a target specific sequence and a universal primer sequence, and other half has other more sequences, a variable length random fragment to provide the size differences for electrophoretic resolution.
  63. 63. Multiplex ligation-dependent probe amplification (MLPA)  A pair of probes is hybridized on the target region adjacently so that they can then be joined by use of a ligase; the contiguous probe can be amplified by PCR  After PCR amplification, the copy number of target sequence i.e. deletion or duplication of target sequence can be determined and quantified using the relative peak heights
  64. 64. Multiplex ligation-dependent probe amplification (MLPA)
  65. 65. Detection of Unknown Mutations Single Strand Conformational Polymorphism (SSCP) Denaturing Gradient Gel Electrophoresis (DGGE) Heteroduplex analysis Restriction fragment length polymorphism (RFLP)
  66. 66. Single Strand Conformational Polymorphism (SSCP)  SSCP is one of the simplest screening techniques for detecting unknown mutations (microlesions) such as unknown single-base substitutions, small deletions, small insertions, or micro-inversions  A DNA variation causes alterations in the conformation of denatured DNA fragments during migration within gel electrophoresis  The logic is comparison of the altered migration of denatured wild-type and mutant fragments during gel electrophoresis
  67. 67. Single Strand Conformational Polymorphism (SSCP)  DNA fragments are denatured, and renatured under special conditions preventing the formation of double-stranded DNA and allowing conformational structures to form in single-stranded fragment  The conformation is unique and resulted from the primary nucleotide sequence  Mobility of these fragments is differed through non- denaturing polyacrylamide gels; detection of variations is based on these conformational structures.  PCR is used to amplify the fragments, called PCR-SSCP, because the optimal fragment size can be 150 to 200 bp.  About 80–90% of potential point mutations are detected by SSCP
  68. 68. Denaturing Gradient Gel Electrophoresis (DGGE):  DGGE has been used for screening of unknown point mutations. It is based on differences in the melting behavior of small DNA fragments (200-700 bp); even a single base substitution can cause such a difference.  In this technique, DNA is first extracted and subjected to denaturing gradient gel electrophoresis.  As the denaturing condition increases, the fragment completely melts to single strands.  The rate of mobility in acrylamide gels depends on the physical shape of the fragment
  69. 69. Denaturing Gradient Gel Electrophoresis (DGGE):  Detection of mutated fragments would be possible by comparing the melting behavior of DNA fragments on denaturing gradient gels.  Approximately less than 100% of point mutations can be detected using DGGE.  Maximum of a nearly 1000 bp fragment can be investigated by this technique
  70. 70. Heteroduplex analysis  A mixture of wild-type and mutant DNA molecules is denatured and renatured to produce heteroduplices  Homoduplices and heteroduplices show different electrophoretic mobilities through nondenaturing polyacrylamide gels  In this technique, fragment size ranges between 200 and 600 bp, Nearly 80% of point mutations have been estimated to be detected by heteroduplex analysis
  71. 71. Restriction fragment length polymorphism (RFLP)  Point mutations can change restriction sites in DNA causing alteration in cleavage by restriction endonucleases which produce fragments with various sizes  RFLP is used to detect mutations occurring in restriction sites
  72. 72. Next Generation Sequencing
  73. 73. Next Generation Sequencing  High speed and throughput, both qualitative and quantitative sequence data are allowed by means of NGS technologies so that genome sequencing projects can be completed in a few days  NGS systems provide several sequencing approaches including whole-genome sequencing (WGS), whole exome sequencing (WES), transcriptome sequencing, methylome, etc.  The coding sequences compromises about 1% (30Mb) of the genome.  More than 95% of the exons are covered by WES; on the other hand, 85% of disease-causing mutations in Mendelian disorders are located in coding regions. Sequencing of the complete coding regions (exome), therefore, could potentially uncover the mutations causing rare, mostly monogenic, genetic disorders as well as predisposing variants in common diseases and cancer.

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