1. 1 Biotechnology and Human Disease (Molecular Basis of Inherited Diseases) Biotechnology: is the use of biological knowledge in the scientific study of technology and vice versa.

2. 2 Outline Restriction endonucleases DNA cloning Probes Southern Blotting Restriction Fragment Length Polymorphism Polymerase chain reaction Analysis of gene expression Gene therapy Transgenic animals Biopharmaceuticals

3. OVERVIEW The entire sequence of the human genome is now known. This effort is called the Human Genome Project, with the help of: Restriction endonucleases: this permits the dissection of huge DNA molecules into defined fragments. Cloning techniques: for amplification of specific nucleotide sequences. Probe: is a fragment of DNA or RNA of variable length (usually 100-1000 bases long) which is radioactively labeled. It can then be used in DNA or RNA samples to detect the presence of nucleotide sequences (the DNA target) that are complementary to the sequence in the probe Synthesis of specific probes: for identification and manipulation of the sequence of interest. These techniques allow the identification of both normal and mutant sequences in DNA, leading to the development of methods for: Prenatal diagnosis of genetic diseases and Treatment of patients by gene therapy. 3

4. RESTRICTION ENDONUCLEASES One of the major obstacles to molecular analysis of genomic DNA is the massive size of the molecules involved. Restriction endonucleases (restriction enzymes): Bacterial enzymes that cleave double-stranded DNA into smaller, more manageable fragments Each enzyme cleaves DNA at a specific palindromic nucleotide sequence (4-6bp), producing restriction fragments. DNA sequence that is recognized by a restriction enzyme is called restriction site These enzymes form either staggered cuts (sticky or cohesive ends) or blunt end cuts on the DNA Bacterial DNA ligases can anneal two DNA fragments from different sources if they have been cut by the same restriction endonuclease  the hybrid combination of two fragments is called a recombinant DNA molecule 4

5. 5 This means, within a short region of the double helix, the sequence on the "top" strand, read 5  3, is identical to that of the "bottom" strand, also read in the 5  3 direction. Specificity of restriction endonucleases

6. "Sticky" and "blunt" ends: – These enzymes cleave DNA to produce a 3-OH group on one end and a 5-phosphate group on the other. – Some of them  form staggered cuts that produce "sticky" or cohesive ends (gives DNA fragments with complementary single-stranded sequences). – Others cleave in the middle of their recognition sequence (i.e., at the axis of symmetry) to produce fragments with "blunt" ends that do not form H- bonds with each other.

7. "Sticky" and "blunt" ends: -Using DNA Ligase, sticky ends of a DNA fragment can be covalently jointed with other DNA fragments with sticky ends produced by the same restriction enzyme. -The hybrid combination of the 2 fragments is a recombinant DNA molecule. -Another Ligase encoded by bacteriophage T4 can covalently join blunt-ended fragments. 7

8. Nomenclature According to the organism from which the enzyme was isolated: The 1st letter of the name from the genus of the bacterium. The next 2 letters (from the type or strain). A final number: to indicate the order in which the enzyme was discovered in that organism. Ex.: HaeIII is the third restriction endonuclease isolated from the bacterium Haemophilus aegyptius. TaqI is the first restriction endonuclease isolated from the bacterium Thermus aquaticus.

9. DNA CLONING is: The introduction of a foreign DNA molecule into a replicating cell for amplification (production of many copies) of the DNA The total cellular DNA is cleaved with a specific restriction enzyme to give 100,000s of fragments 1.Each fragment is ligated to a DNA vector (cloning vector) to form a hybrid molecule. 2.Hybrid recombinant DNA molecule transfers its DNA fragment into a single host cell (ex: bacterium) for replication (amplification). 3.Then the host cell multiplies forming a clone in which each cell carries copies of the same inserted DNA fragment (cloning). 4.The cloned DNA is released from its vector by cleavage using the appropriate restriction endonuclease, then isolation 9

10. (a) DNA recombination. (b) Transformation. (c) Selective amplification. (d) Isolation of desired DNA clones. The DNA fragment to be cloned is inserted into a vector with antibiotic resist gene The recombinant DNA enters into the host cell and proliferates. A specific antibiotic is added to kill E. coli without any protection. The transformed E. coli is protected by the antibiotic- resistance gene whose product can inactivate the specific antibiotic. antibiotic- resistance gene 10 Biotechnology CH1. f

11. Vectors:  A vector is a molecule of DNA to which the fragment of DNA to be cloned is joined.  Commonly used vectors: plasmids. and bacterial and animal viruses  Essential properties of a vector include: It must be capable of autonomous (independent) replication within a host cell. It must contain at least one specific nucleotide sequence recognized by a restriction endonuclease. It must carry at least one gene that confers the ability to select for the vector such as an antibiotic resistance gene. 11

12. Prokaryotic plasmids : The small, circular, extra chromosome DNA. Plasmid may: – carry antibiotic resistance genes – facilitate the transfer of genetic information from one bacterium to another. – be isolated from bacterial cells and cleaved at specific sites by restriction endonucleases, and foreign DNA is inserted. -The hybrid plasmid can be reintroduced into a bacterium for amplification (large number of copies of the plasmid containing the foreign DNA) 12

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14. DNA libraries:  A DNA library is a collection of cloned restriction fragments of the DNA of an organism.  There are two kinds of libraries: genomic libraries and (complementary) cDNA libraries.  Genomic libraries contain a copy of every DNA nucleotide sequence in the genome, while cDNA libraries contain DNA sequences that appear as mRNA molecules and these differ from one cell type to another.  Cloned cDNAs lack introns and the control regions of the genes, whereas these are present in genomic libraries. 14

15. DNA libraries:

16. Making a DNA library A DNA library consists of DNA from a particular organism inserted into bacterial plasmids. 16

17. Complementary DNA (cDNA) libraries: Using mRNA as a template to make a complementary double-stranded DNA (cDNA) using the enzyme Reverse Transcriptase. The resulting cDNA is a double- stranded copy of mRNA. cDNA can be amplified by cloning or by polymerase chain reaction. 17

18. cDNA libraries If the mRNA used as a template in a mixture of different species, the resulting cDNAs are heterogenous and cloned to form a cDNA library. cDNA has no intervening sequences (introns and control regions), it can be cloned into an expression vector for the synthesis of eukaryotic proteins by bacteria. 18 Expression vectors are plasmids containing a bacterial promoter for transcription of the cDNA, and a Shine-Dalgarno sequence for translation of the resulting mRNA by bacterial ribosome.

19. How to produce a recombinant protein 10 to 70% of cellular protein 0.1 to 1% of cellular protein

20. Gel Electrophoresis When DNA is treated with restriction enzymes, the DNA is cut into fragments of various sizes. These fragments can be separated in a gel on the bases on their electric charge and size. 1. DNA is cut up with restriction enzymes. 2. The DNA fragments are transferred to wells cut into a gel. 3.An electric charge is transferred through the gel. The negatively charged DNA fragments are repelled by the negative electrode and attracted to the positive electrode. The samples are run at the same time, in the same gel. The resulting pattern is known as a DNA fingerprint. If identical patterns appear in different column the samples may be from the same source 20 Biotechnology CH1. f

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22. Ethidium bromide-stained products after gel electrophoresis.gel electrophoresis Ladder 22

23. 23 Complementary DNA (cDNA) libraries: Using mRNA as a template to make a complementary double-stranded DNA (cDNA) using the enzyme Reverse Transcriptase. The resulting cDNA is a double-stranded copy of mRNA. cDNA can be amplified by cloning or by polymerase chain reaction. It can be used as a probe to locate the gene that coded for the original mRNA (or fragments of the gene) in mixtures containing many unrelated DNA fragments.

24. Sequencing of cloned DNA fragments: Sanger dideoxy method: is a procedure used to determine the base sequence of DNA fragments that have been cloned and purified. Procedure: 1.The single-stranded DNA (ssDNA) to be sequenced is used as the template for DNA synthesis by DNA polymerase. 2.A radioactive primer complementary to the 3-end of the target DNA is added along with the four deoxyribonucleoside triphosphates (dNTPs). 3.Divide the sample into 4 rxn tubes and add to each one a small amount of one of the 4 dideoxy ribonucleoside triphosphate (ddNTPs). Because ddNTPs contain no 3’-OH group, incorporation of a ddNTPs into a newly synthesized strand terminates its elongation. 4.The products of this reaction is a mixture of DNA strands of different lengths, each terminating at a specific base. 5.Separation of DNA products by size using polyacrylamide gel electrophoresis 6.Autoradiography; yields a pattern of bands from which the DNA sequence can be read. 24

25. Dideoxynucleotides DNA Sequencing using the Sanger method involves the use of 2’3’- dideoxynucleotide triphosphates in addition to regular 2’-deoxy nucleotide triphosphates (ddNTPs and dNTPs) 2’3’-dideoxynucleotide triphosphates lack a 3’ OH group, and DNA polymerization occurs only in the 3’ direction, once 2’3’- dideoxynucleotide triphosphates are incorporated, primer extension stops 25 H P O OH HO O O CH 2 NH 2 N N N N Sugar Base Phosphate 3’ 5’ 2’ 1’4’ H 2’3’-dideoxynucleotide monophosphate 2’-dideoxynucleotide monophosphate

26. 2’3’ dideoxy- nucleotides Terminate DNA Replication 26 SUGAR-PHOSPHATE BACKBONE H P O HO O O CH 2 HOH P O O HO O O CH 2 H P O OH HO O O CH 2 NH 2 N N N N O O N NH N N N O NH 2 N B A S E S O H P O HO O O CH 2 HO O H2NH2N N HN N N H HOH P O O O CH 2 CH 3 O O HN N OH H P HO O O CH 2 H O N O H2NH2N N H2OH2O 2’3’dideoxynucleotide

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28. DNA Sequencing In DNA sequencing reactions all the basic components needed to replicate DNA are used 4 reactions are set up, each containing: – DNA Polymerase – Primer – Template to be sequenced – dNTPs – A small amount of one ddNTP ddATP, ddCTP, ddGTP, ddTTP As incorporation of ddNTPs terminates DNA replication, a series of fragments is produced all terminating with the ddNTP that was added to each reaction 28

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30. DNA Sequencing What A Sequencing Autorad Actually Looks Like To read the autorad it is important to start at the bottom and work up so that it is read in the 5’ to 3’ direction 30 A C G T 5’ CTAGAGGATCCCCGGGTACCGAGCT... 3’ Biotechnology CH1. f

31. PROBES Probe: is a single-stranded piece of DNA, labeled with a radioisotope (e.g. 32 P) or with a non-radioactive probe, (e.g. biotin). The nucleotide sequence of a probe is complementary to the target DNA. Probes are used to identify which clone of a library or which band on a gel contains the target DNA. Hybridization of a probe to DNA fragments: The utility of probes depends on the phenomenon of hybridization. Hybridization: is a process in which a single-stranded sequence of a target DNA binds to a probe of a complementary nucleotide sequence (DNA probe-target DNA hybrid duplex) 31

33. Hybridization of a probe to DNA fragments 1)ssDNA is produced by alkaline denaturation of dsDNA. 2)This ssDNA is first bound to a nitrocellulose membrane to prevent self-annealing. (DNA immobilization). 3)The immobilized DNA strands are available for hybridization to an exogenous, single-stranded complementary, radio labeled DNA probe. 4)The extent of hybridization is measured by the retention of radioactivity on the membrane. 5)Excess probe molecules that do not hybridize are removed by washing the filter and so do not interfere. 33

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35. Detecting the ß S -globin mutation Allele-specific oligonucleotide (ASO) is a short piece of synthetic DNA complementary to the sequence of a variable target DNA. It acts as a probe for the presence of the target DNA  probe can be used to  detect the presence of the sickle cell mutation in the ß-globin gene. 1. DNA, isolated from white blood cells, is denatured into single strands. 2. An oligonucleotide is constructed that is  complementary to the portion of the mutant globin gene coding for the amino- terminal sequence of the ß-globin protein. 3.Thus, a double-stranded hybrid forms that can be detected by electrophoresis. 35Biotechnology CH1. f

36. Detecting the ß S -globin mutation DNA isolated from a heterozygous individual (sickle cell trait) or a homozygous patient (sickle cell disease) contains a nucleotide sequence that is complementary to the probe DNA obtained from normal individuals is not complementary at the sixth codon (coding for glutamate in normal individuals but for valine in patients with the ß S -gene) and, therefore, does not form a hybrid 36 Biotechnology CH1. f

37. Biotinylated probes Because the expensive disposal of radioactive waste,  non-radioactive probes have been developed. The vitamin biotin, can be chemically coupled to the nucleotides used to synthesize the probe. Biotin binds very tenaciously to avidin  a readily available protein contained in chicken egg whites Avidin can be attached to a fluorescent dye detectable optically with great sensitivity. Thus, a DNA fragment (displayed by gel electrophoresis) that hybridizes with the biotinylated probe is made visible by  immersing the gel in a solution of dye- coupled avidin. After washing away the excess avidin, the DNA fragment that binds the probe is fluorescent. 37

38. SOUTHERN BLOTTING A technique that detects mutations in DNA. It combines the use of restriction enzymes and DNA probes. Named after its inventor Edwards Southern Procedure (fragmentation  electrophoresis  detection) 1.DNA (entire genomic) is extracted from leukocytes and cleaved using a specific restriction enzyme  million of fragments 2.Resulting fragments are separated on the basis of size by electrophoresis (larger is slower, smaller is faster) and their lengths are determined in bp 3.DNA fragments in gel are denatured and transferred to nitrocellulose membrane 4.A specific probe hybridization helps to visualize DNA fragments of interest 38

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41. RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) Genome variations are  differences in the sequence of DNA among individuals. They include both: polymorphisms and mutations. A polymorphism is a clinically harmless DNA variation. It often occurs in the  intervening sequences that do not code for proteins Mutation refers to an infrequent potentially harmful genome variation that is  associated with a specific human disease. A restriction fragment length polymorphism (RFLP): is a genetic variant that can be examined by cleaving the DNA into fragments (restriction fragments) with a restriction enzyme. The length of the restriction fragments is altered if the genetic variant alters the DNA so as to create or abolish a restriction site  In either case, cleavage with an endonuclease results in fragments of lengths differing from the normal ( more or fewer), which can be detected by DNA hybridization 41

42. Polymerase Chain Reaction Polymerase chain reaction (PCR) : enables researchers to produce millions of copies of a specific DNA sequence in approximately two hours. This automated process bypasses the need to use bacteria for amplifying DNA. The name, polymerase chain reaction, comes from the DNA polymerase used to amplify (replicate many times) a piece of DNA by in vitro enzymatic replication. The original molecule or molecules of DNA are replicated by the DNA polymerase enzyme, thus doubling the number of DNA molecules. Then each of these molecules is replicated in a second "cycle" of replication, resulting in four times the number of the original molecules. Again, each of these molecules is replicated in a third cycle of replication. This process is known as a "chain reaction" in which the original DNA template is exponentially amplified. Biotechnology CH1. f

43. POLYMERASE CHAIN REACTION(PCR) PCR is a test tube method for amplifying a selected DNA sequence that does not rely on the biologic cloning method. PCR permits the synthesis of millions of identical copies of a specific nucleotide sequence in a few hours. The method can be used to amplify DNA sequences from any source-bacterial, viral, plant, or animal. PCR is an artificial way of doing DNA replication. PCR uses DNA polymerase to repetitively amplify target DNA Each cycle of amplification doubles the amount of DNA in the sample, leading to an exponential increase in DNA with repeated cycles of amplification

44. To perform a typical PCR reaction, some sequence information about two regions of your DNA of interest is required so that appropriate primers may be synthesized. The two primers, each typically about 15-20 nucleotides in length, are usually designed so they are 200-2000 bp apart, one hybridizing to one strand of dsDNA, the other hybridizing to the other strand such that both primers are oriented with their 3' ends pointing towards each other. 44Biotechnology CH1. f

45. H:\Biochemistry course\bio 2 all\DNA animations\PCR Virtual Lab\PCRVirtualLab.html 45Biotechnology CH1. f

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47. How The Functions Of Replication Are Achieved During PCR è N/A as fragments are short Joining nicks è Taq DNA PolymerasePolymerizing DNA è Primers are added to the reaction mix Providing primer PCRFunction è Heat Melting DNA Components of a PCR Reaction Buffer (containing Mg ++ ) Template DNA 2 Primers that flank the fragment of DNA to be amplified dNTPs Taq DNA Polymerase (or another thermally stable DNA polymerase)

48. Steps of a PCR 1.Primer construction: synthetic oligonucleotide (20-35 NMP) single stranded and complementary to the Flanking sequences -nucleotide sequence on each side of the target DNA.The 3'-hydroxyl end of each primer points toward the target sequence. 2.Denature the DNA: The DNA to be amplified is heated to separate the double-stranded target DNA into single strands. 3.Annealing of primers to single-stranded DNA: The separated strands are cooled and allowed to anneal to the two primers (one for each strand). 4.Chain extension: DNA polymerase adds deoxyribonucleotides (within reaction mixture in excess) to the 3'-hydroxyl end of the primer, and strand growth –complementary antiparallel- extends across the target DNA. 48Biotechnology CH1. f

49. Applications of PCR 1.Comparison of a normal cloned gene with an uncloned mutant form of the gene 2.Detection of low-abundance nucleic acid sequences 3.Forensic analysis of DNA samples 4.Prenatal diagnosis and carrier detection of cystic fibrosis 49Biotechnology CH1. f

50. ANALYSIS OF GENE EXPRESSION Products of gene expression are mRNA and proteins. A. Determination of mRNA levels Northern blots: The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample (mRNAs electrophoresis  transfer to a membrane  hybridization to a radioactive probe)

51. Microarrays are used to determine the differing patterns of gene expression in normal and cancer cells DNA microarrays contain thousands of immobilized DNA sequences in tiny spots (1000s, each corresponding to a different gene) organized in a microscope slide (gene chip) Used for genotyping (of genomic DNA variations or mutations) or gene expression analysis (mRNA converted to cDNA coupled to florescent tag) Fluorescence bound to each spot is a measure of the amount of corresponding mRNA in sample

52. B. Analysis of proteins Proteomics is the study of all proteins expressed by a genome, including their relative abundance, distribution, posttranslational modifications, functions, and interactions with other macromolecules. Enzyme linked immunosorbent assay (ELISA) uses labeled antibodies (probe) in sandwich technique to detect and quantify specific proteins in multiwell (microtiter) plate – Antigen (protein) is bound to wells – Enzyme-coupled antibodies are specific for this particular antigen, and produce a colored product when exposed to substrate – Amount of color is used to determine amount of protein (antigen) in sample

53. Western Blots (immunoblots) – Western blot analysis of proteins separated by electrophoresis. – The Western blot is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. – It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non- denaturing conditions). – The proteins are then transferred to a membrane (typically nitrocellulose), where they are probed (detected) using antibodies specific to the target protein 53Biotechnology CH1. f

55. GENE THERAPY Gene replacement therapy is to insert the normal, cloned DNA for a gene into the somatic cells of a patient who is defective in that gene as a result of some disease- causing mutation The DNA must become permanently integrated into the patient's chromosomes to be expressed to produce the correct protein. 55

56. TRANSGENIC ANIMALS Transgenic animals can be produced by injecting a cloned gene into the fertilized egg. If the gene becomes successfully integrated into a chromosome, it will be present in the germline of the resulting animal, and can be passed along from generation to generation. Transgenic goats and cows can now be designed to produce human hormones in their milk. Sometimes, rather than introducing a functional gene into a transgenic mouse, a mutant gene is used to replace the normal copies of that gene in the cells of the mouse. This can be used to produce a colony of "knockout mice" that are deficient in a particular enzyme. Such animals can then serve as models for the study of a corresponding human disease.

57. Biopharmaceuticals Are recombinant therapeutic proteins, monoclonal antibodies products used in vivo and nucleic acid- based medicinal products Represent 1 in every 4 new pharmaceuticals (new molecular entities) Therapeutic goals of biopharmaceutical engineering – The reduction/elimination of product immunogenecity – The generation of products with altered pharmacokinetic profiles – The alteration of biological half life – The generation of novel (hybrid) proteins 57

58. Gene: is a segment of DNA that codes for a specific polypeptide Genome: is the genetic information content of a cell. Hormone is an endogenous substance that is secreted by one type of cell and acts on another type of cell. Hybridoma: is a hybrid cell produced by the fusion of a myeloma cell and a specific antibodyproducing B lymphocyte. A single hybridoma produces a single type of antibody. Interferon is any of a class of glycoproteins produced by animal cells in response to viral infection. Interleukin is a group of proteins synthesized by macrophages and T lymphocytes in response to antigen and other stimulation. Lymphokine is any of a class of soluble proteins produced by some white blood cells. These proteins stimulate other white blood cells as part of the immune response. Tumor necrosis factor (TNF) is a lymphokines produced by macrophage. It can be activated to kill tumor

59. Antigen is a substance that stimulate production of antibody Antibody is an immunoglobulin produced by the body in response to stimulation from an antigen. Antisense DNA is a complementary strand of DNA that is specifically synthesized to attach to the sense DNA and prevent genetic transcription. The sense DNA that carries the information that affects the disease process is usually elucidated before an antisense drug is designed. Colony-stimulating factors (CSFs) are a class of glycoprotein hormones. CSFs regulate the differentiation and formation of blood cells from precursor cells. Cytokines are a group of special proteins (nonantibodies) released by cells to trigger action in other cells.