Recombinant DNA Technology in Medicine PDF

Gene Cloning and Recombinant DNA Technology in Medicine Prof. Bart Dzudzor  Recombinant DNA Technology Objectives: Involves techniques in manipulating DNA:- Molecular Cloning. DNA sequencing. Polymerase Chain Reaction (PCR). Nucleic acid blotting and hybridization. (Southern, N...

Gene Cloning and Recombinant DNA Technology in Medicine Prof. Bart Dzudzor  Recombinant DNA Technology Objectives: Involves techniques in manipulating DNA:- Molecular Cloning. DNA sequencing. Polymerase Chain Reaction (PCR). Nucleic acid blotting and hybridization. (Southern, Northern analysis). Production of proteins. Creation of Knock-out, Knock-in and Transgenic mice. Nucleic acid Microarray (simultaneous monitoring of expression level of each gene in a cell.  Restriction Endonucleases Their discovery revolutionized recombinant DNA technology because they can cut DNA double helix at specific sites defined by their local nucleotide sequence. They are purified from bacteria. These enzymes recognize short sequences of DNA usually 4-8bp long and cut both strands. These sequences, where they occur in the genome of the bacterium itself, are protected from cleavage by methylation at an A or a C residue; the sequences in foreign DNA are generally not methylated and so are cleaved by the restriction enzymes. The recognition sequences are usually palindromes-that is, the sequence is the same if read from one direction on one strand and the opposite direction on the other strand. Some restriction enzymes produce staggered cuts ( leaving what are known as “sticky ends” or “cohesive”) and others produce blunt cuts (leaving “blunt ends”). The fragments of DNA produced by cleavage with a restriction enzyme are known as restriction fragments. In the case of an enzyme that produces sticky ends, the ends can transiently base- pair. The base-paired ends can be covalently joined by the action of DNA ligase. Restriction-recognition sites are short DNA sequences recognized and cleaved by various restriction endonucleases. Restriction Enzymes are used to digest (cut) fragments of DNA for the purposes of: fractionating genomic DNA by size (Southern blot). cloning a specific sequence (generate library). mapping a region of DNA (restriction site map). isolating a specific fragment of DNA (to make a labeled probe).  RESTRICTION FRAGMENT Cut the DNA with RE How often do they cut? → If the DNA sequence is completely random (it’s not), the probability of a sequence occuring is: 6 bp recognition sequence : e.g. EcoRI (1/4)6 = 1/4096 ~ 1 cut every 4 kb 8 bp “ “ (1/4)8 ~ 1 “ “ 65 kb 4 bp “ “ (1/4)4 ~ 1 “ “ 250 bases Have isolated >3000 Type II RE with >200 sequence specificities. Isolate the desired DNA fragment. Usually done by gel electrophoresis on agarose gels (non-denaturing).  Why do Medical Researchers Clone Genes and how does that contribute to the field of Medicine? A. To define inherited genetic mutations that cause/predispose to disease making it possible to: i. develop diagnostic reagents. ii. Develop pre-natal testing and counseling. iii. Define the precise biochemical defect. iv. Develop therapies that treat the disease instead of the symptoms; a target therapy repairs/overcomes the inherited defect.  B. To isolate functional (“normal”) genes that can be harnessed to produce therapeutic molecules a. Proteins produced by recombinant DNA are made outside the body and not contaminated with infectious blood products such as Hepatitis, HIV, etc. For example the clotting factor Factor VIII needed by hemophiliacs. b. Proteins may be used not merely to replace a low level or nonfunctional protein, but also generate a desired effect in persons with a functional gene. For example, certain blood cell growth factors are given to allow harvest of bone marrow progenitor cells from peripheral blood. c. Genes can be isolated from infectious organism to allow vaccine development. Eliminates the possibility of reversion to a wild-type from an attenuated organism.  C. Isolate genes which contain somatic mutations (not inherited but acquired in a specific cell population) a. Develop a diagnostic for certain rearrangements or mutations in tumors that leads to the selection of a specific type of therapy. b. Use as a marker to measure response to therapy or disease progression. c. Target specific therapies to recognize the altered gene. i. antibodies against surface proteins ii. Tyrosine kinase inhibitors iii. Gene therapy iv. Stimulate the immune system. D. To obtain an enhanced understanding of biochemical pathways in order to expand our knowledge base (signal transduction, immunity, development, the cell cycle) A. Integrate the discovery of a new gene into the existing pathway to make predictions about its function. B. “knowledge is power” basic research Clinical research New modalities of patient care.  Molecular Cloning. To study and manipulate a particular DNA sequence or gene, it is usually necessary to amplify it. The amplification process is called cloning (more precisely-molecular cloning) since a single recombinant DNA molecule is replicated into million of identical copies. This is different from cloning organisms which is the process of taking a single cell from an organism and using it to regenerate an identical copy of the organism. Molecular cloning involves joining a specific DNA fragment to a DNA molecule that can replicate in a host cell-usually the bacterium E. coli. The replicating DNA molecule is called a vector. Since the vector can replicate in the bacterial cells, any heterologous (foreign) DNA covalently joined to the vector will also be replicated. Common vectors are Plasmids and bacterial viruses. In eukaryotic cells, vectors can also be either plasmids or viruses (retroviruses).  The important features to consider when choosing a vector are: should be small and easy to handle. able to generate large amounts of DNA through replication in an appropriate organism such as bacteria. if necessary can direct expression of a recombinant protein in appropriate host cell. can accommodate DNA of the required size. has an appropriate selection marker (antibiotic resistance) PLASMID CLONING VECTOR A plasmid is an extrachromosomal double- stranded DNA molecule that replicates autonomously in bacterial cells General procedure for cloning a DNA fragment in a plasmid vector. B A Isolation of DNA fragments from a mixture The plasmid DNA can then be isolated from cells in the colony and characterized or by cloning in a plasmid vector. otherwise manipulated.  PLASMIDS A plasmid is an extrachromosomal double-stranded DNA molecule that replicates autonomously in bacterial cells  PLAMID VECTORS Plasmids used for DNA cloning usually have been engineered to contain: a number of convenient restriction sites a marker gene to select for its presence in the host cell Simple fusion of different DNA fragments EcoRI EcoRI pBR322, cut at its single Eco RI site DNA ligase EcoRI foreign EcoRI recombinant DNA DNA Sticky ends New Eco RI site NNNGAATT CNNN NNNGAATTCNNN NNNC TTAAGNNN NNNCTTAAGNNN EcoRI end on insert EcoRI end on vector Fusion site pUC19 Polylinker: restriction sites lacZ+ gene Origin Ampicillin sequence resistance gene Cloning of cDNA using BamHI linkers Alternative: use adapter 5’-GATCCAGAC-3’ GTCTG-5’  BLUE/WHITE SELECTION Blue-white selection is used to identify colonies which have insert-containing plasmids – eliminates those that have plasmids with no DNA insert Use a pUC cloning vectors and host cells which produce an incomplete (inactive) -galactosidase protein MCS in pUC vector is in the middle of the LacZ gene The blue-white selection involves addition of IPTG and X-gal to the medium IPTG induces the LacZ operon  BLUE/WHITE SELECTION The LacZ operon results in expression of the - galactosidase -peptide The -galactosidase -peptide complements the incomplete (inactive) -galactosidase protein in the host E. coli cells and produces functional , active - galactosidase Functional -galactosidase cleaves the colourless X-gal in the medium to give active colonies a blue colour. IF a plasmid contains a DNA insert in the MCS (i.e. in the middle of the LacZ gene), then a functional - peptide cannot be generated, complementation does not occur, and colonies cannot cleave X-gal. Therefore, colonies with plasmids with inserts stay white.  Some uses of cloning: 1. Isolate and characterize a specific gene or mRNA from a complex mixture (the genome or the total RNA in a cell). 2. Generate probes to detect homologous sequences-useful in disease diagnosis. 3. Express large amounts of protein-useful in producing therapeutic proteins. 4. Generate mutations to develop animal models of particular diseases.  DNA Libraries Libraries are a complex collection of DNA fragments (for example, the DNA fragments produced by cutting human DNA with a restriction fragment) individually inserted into a vector. Genomic Libraries Genomic libraries consist of DNA derived from the genome of an organism. Genomic libraries therefore contain all elements of the genome-coding regions, intron, regulatory control regions, regions between gene, etc.  cDNA Libraries cDNA libraries contain double-stranded copies of DNA copied from mRNA. cDNA library only contains the sequences that are present in the mRNAs. It also contains only those sequences that are expressed-therefore two cDNA libraries made from two cell types in an organism will have overlapping but distinct sets of sequences. cDNA is synthesized using reverse-transcriptase-an enzyme that synthesizes a single strand DNA copy of an mRNA. The single stranded DNA can then be converted into a double-stranded molecule using a DNA polymerase and then cloned into a vector. Construction of a human Genomic DNA library.  Genomic library contains all of the genetic information found in the genome of an organism. In the case of human DNA introns, exons and noncoding regions would all be included. Reasons to construct and screen a genomic library include: 1. to clone the whole gene including introns and 5’ regulatory sequences (promoter), for example to study gene expression. 2. the gene will be cloned in the context of surrounding DNA making it possible to move along a chromosome and clone a new gene whose approximate chromosomal location is known. 3. to make knock-out animal model by replacing the functional gene with a nonfunctional copy. The Synthesis of cDNA When thinking about constructing or using a cDNA library remember: There are no upstream regulatory sequences controlling gene expression because they are not transcribed into the mRNA. The cellular source of mRNA is critical! If the cell does not express the gene , no mRNA, no cDNA and it won’t be in the library. Since there are no introns, cDNA clones can be “translated” to find open reading frames and predict protein sequence. It is not possible to determine chromosomal “context” or identify relationship to genes that map nearby. The differences between cDNA clones and genomic DNA clones derived from the same region of DNA  Nucleic Acid Hybridization A common method to detect a specific nucleotide sequence in a complex mixture such as a library is hybridization. Hybridization is based on the ability of a single- stranded nucleic acid (probe) to specifically anneal by base pairing to a complementary sequence present among many copies of non- complementary sequences. To detect the hybridization product, the probe is modified so that it can be detected-for example with radioactive or fluorescent nucleotides. Membrane hybridization assay for detecting nucleic acids. This assay can be used to detect both DNA and RNA, and the radiolabeled complementary probe can be either DNA or RNA.  Southern and Northern Blotting These are procedures in which mixtures of nucleic acids are separated by size and then probed by hybridization. To perform a southern blot, the genomic DNA is first digested to completion with one or more restriction endonucleases. The size separation is carried out by gel electrophoresis in which DNA or RNA is applied to a porous gel and then subjected to an electric field. The negatively charged DNA or RNA migrates towards the positive electrode. The smallest fragments migrate the fastest and therefore at the bottom of the gel. The molecules are then transferred to a solid support such as nitrocellulose filter. Before the transfer, the DNA gel is treated with an alkaline solution to denature the DNA, this separates the two complementary strands making them available for hybridization with the probe. The blot is then hybridized to a labeled DNA probe (can be either radioactive or non radioactive system) to detect the gene or RNA of interest. Southern blot technique for detecting the presence of specific DNA sequences In Northern blotting, the total cellular RNA is denatured by treatment with an agent (e.g formaldehyde) that prevents H-bonding between base pairs, ensuring that all the RNA molecules have unfolded, linear conformation.  Southern Blot can be used to Detect: large insertion or deletion (50 to 100bp) of chromosomal DNA into the gene; the restriction fragments which hybridize to the probe will be either larger or smaller than expected. gross gene amplification, this means that rather than the normal number of 2 copies per diploid cell there may be 5 to 20 or more copies per cell. (such as the HER-2/neu gene which plays a causative role in the development of a highly aggressive breast cancer). genomic rearrangements caused by chromosomal translocation. This occurs when two chromosomes actually break and re-join with the wrong chromosome (an example is the BCR-Abl translocation that plays a causative role in the development of chronic myeloid leukemia (CML)). mutation of a single nucleotide can only be seen on a Southern blot if it creates or destroys a restriction enzyme site (Hemophilia A and sickle cell diseases are examples).  A Southern Blot does not detect: whether or not a gene is expressed point mutations (other than restriction fragment length polymorphism) small deletions.  A Northern Blot can detect: expression of a specific gene which likely changes from tissue to tissue (except for “housekeeping” genes). the size of the RNA transcript. An aberrant sized mRNA could arise as a result of deletion or insertion in the gene or a chromosomal translocation. the relative levels of expression in different samples. Increased or decreased levels of mRNA are associated with many diseases states northern can detect deletions or insertions in genes as well as splicing defects.  Polymerase Chain Reaction Procedure that can amplify a specific sequence without cloning into a vector. It is very useful in clinical and forensic settings where limited amounts of DNA are available. Use short, chemically-synthesized primers that flank region to be amplified. Use enzyme from bacteria that lives at high temperature such as Thermus aquaticus. Template is melted at high temperature and annealed to excess amounts of primers at lower temperatures. Copies are made by the polymerase using dNTPs. The cycle is repeated about 20 to 40 cycles. Since each round results in a replication cycle, if carry out 20 cycles will get 220= 1,048,576 copies.  PCR continued. Because of the amplification properties of PCR it is a very powerful technique to detect very low levels of DNA or RNA. For example it is used as a very sensitive test for HIV infection since PCR can be used to amplify sequences from the RNA genome of the virus. It is also sensitive enough to detect specific sequences in small amounts of material available from crime scenes. It is so sensitive that a fingerprint or saliva left on the back of a stamp contains enough DNA for analysis by PCR. PCR is very fast compared to Southern or Northern blotting so it has replaced these techniques in many areas. PCR technique PCR The Key Steps in the PCR reaction are: 1. denature the template DNA by heating to 94o for 5 minutes. 2. anneal the specific primer by lowering the temperature to around 50o. 3. extend the DNA strand using DNA polymerase (Taq) for 5 minutes (72-75o). 4. heat 20 seconds to separate the newly synthesized short strands. 5. go to step 2 and repeat 25 to 40 cycles.  Uses of PCR To look for mutations either inherited or acquired, by doing a PCR reaction followed by DNA sequencing. To detect Minimal Residual Disease in leukemia patients using primers made to recognize DNA sequence on either side of the chromosomal breakpoint. The presence of a small number of leukemic cells in the midst of a proportionally larger number of normal cells is detected by the presence of a positive signal. PCR may be used to determine the gender of embryonic cells generated by in vitro fertilization in families carrying X-linked mutations. It can also be used for in vitro mutagenesis to test the effect of a specific mutation on the function of the gene product.  Uses of PCR Cont’d Gene cloning. Carrier screening. Clinical diagnosis/confirmation. Newborn screening. Presymptomatic diagnosis/predisposition screening. Transplant engraftment-distinguishing donor from recipient cells after bone marrow or organ transplant. Parentage/paternity testing-excluding or not excluding an alleged father (or mother). Forensic identification-matching a suspect’s DNA to that found in some crime evidence in order to solve cases of murder, rape, mail fraud, etc.; identifying victims of mass disasters (bombings, airline crashes, wars, genocides).  PCR uses continued Twin zygosity-distinguishing mono-from dizygotic twins (since only monozygotic twins should match at all polymorphic loci tested). Early detection of HIV, the virus that causes AIDS. Surgical specimen mix-ups-to match a mislabeled or unlabeled biopsy specimen to the patient from which it came. DNA fingerprinting Prenatal diagnosis-several sample types are possible, depending on the clinical situation: Amniocentesis Chorionic villus samples (CVS) Embryonic blastomeres for preimplantation diagnosis Fetal cells circulating in maternal blood. How PCR is used in Forensic Science Three suspects A,B and C, producing 6 DNA bands for each person after PAGE. The band pattern therefore save as a “fingerprint” to identify an individual nearly uniquely. Use of PCR to obtain a Genomic or cDNA clone  Types of DNA Polymorphism RFLP=Restriction Fragment Length Polymorphism-restriction endonuclease recognition sites that are present in some people but not in others (can be detected by Southern blotting or PCR). VNTR=Variable Number of Tandem Repeats-strings of nucleotide sequences (usually of 16 or more nucleotides each), of different repeat number in different people, existing between fixed restriction endonuclease sites; also called minisatellites. STR= Short Tandem Repeats-string of oligonucleotide sequences that are shorter that VNTR (usually of 2,3,4 or 8 nucleotides each), of different repeat number in different people, not necessarily lying between restriction endonuclease sites; also called microsatellites or Simple Sequence Repeats (SSR). The more highly related two individuals are, the more likely it is that a given SSR will be present in the same numbers. SSR variations can be detected by PCR, using primers that lie outside of the repeat region followed by accurate gel electrophoresis of the products. The size of the PCR product then reflects the number of repeats.  Polymorphism cont’d SNP=single Nucleotide Polymorphism-single nucleotide differences between individuals that are not associated with restriction endonuclease sites. Best studied by PCR followed by accurate gel electrophoresis of the products. HLA=the histocompatibility family of genes located on chromosome 6p; although not “junk” DNA, these genes are naturally so highly polymorphic that they can be used for identity studies at either the DNA level or using antibodies to distinguish varieties in the encoded proteins. An example of SSR inheritance  Sanger (Dideoxy) Sequencing Method. Also called dideoxy sequencing because it involves use of 2’,3’-dideoxynucleoside triphosphates (ddNTPs), which lacks a 3’-hydroxy group. In this method, the single-stranded DNA to be sequenced serves as the template strand for in vitro DNA synthesis, a synthetic 5’-end labeled oligodeoxynucleotide is used as the primer. Four separate polymerization reactions are performed, each with a low (100 lower) concentration of one of the 4 ddNTPs in addition to higher concentrations of the normal deoxynucleotide triphosphates (dNTPs)..  Sanger’s Method cont’d In each reaction, the ddNTP is randomly incorporated at the positions of the corresponding dNTP; such addition of a ddNTP terminates polymerization because the absence of a 3’ hydroxy prevents addition of the next nucleotide. The mixture of terminated fragments from each of the four reactions is subjected to gel electrophoresis in parallel; the separated fragments then are detected by autoradiography. The sequence of the original DNA template strand can be read directly from the resulting autoradiogram. Sanger’s (dideoxy) Method for Sequencing DNA fragments Used more frequently than the Maxam-Gilbert method. can be used to sequence longer fragments of DNA than the Maxam-Gilbert method  The enzymatic—or dideoxy— method of sequencing DNA. In each lane, the bands represent fragments that have terminated at a given nucleotide This reaction mixture will eventually produce a set (e.g., A in the of DNAs of different lengths complementary to the leftmost lane) but template DNA that is being sequenced and at different positions terminating at each of the different A’s. The exact in the DNA lengths of the DNA synthesis products can then be used to determine the position of each A in the growing chain.  Strategy for automating DNA sequencing reactions. * Each dideoxynucleotide used in the Sanger method can be linked to a fluorescent molecule that gives all the fragments terminating in that nucleotide a particular color. All four labeled ddNTPs are added to a single tube. The resulting colored DNA fragments are then separated by size in a single electrophoretic gel contained in a capillary tube (a refinement of gel electrophoresis that allows for faster separations). All fragments of a given length migrate through the capillary gel in a single peak, and the color associated with each peak is detected using a laser beam. The DNA sequence is read by determining the sequence of colors in the peaks as they pass the detector. This information is fed directly to a computer, which determines the sequence. Fluorescent Automated DNA sequencing  Production of High levels of Proteins from Cloned cDNA Once the desired cDNA is cloned, large amounts of the encoded protein can be synthesized in engineered E. coli cells. Example, granulocyte colony-stimulating factor can be produced at high levels in expression vectors designed to produce full length proteins at high levels. A simple E. coli expression vector utilizing the lac operon The lacZ gene can be replaced with the G-CSF. When the resulting plasmid is transformed into E. coli cells, addition of IPTG and subsequent transcription from the lac promoter produces G-CSF mRNA, which is translated into G-CSF protein. Two-step expression vector system based on bacteriophage T7 RNA polymerase and T7 late promoter Creation of a Knockout mice Isolation of mouse embryonic stem (ES) cells with a gene targeted disruption by positive and negative selection. When exogenous DNA is introduced into ES cells, random insertion via nonhomologous recombination occurs more frequently than gene-targeted insertion via homologous recombination. Recombinant cells in which one copy of the gene X is disrupted can be obtained by using a recombinant vector that carries gene X disrupted with neor, a neomycin-resistance gene, and outside the region of homology, tkHSV, the thymidine kinase gene from herpes simplex virus Nonhomologous insertions includes the tkHSV gene, whereas homologous insertions doesn’t; therefore, only cells with nonhomologous insertion are sensitive to ganciclovir.  General procedure for producing gene- targeted knockout mice Embryonic stem (ES) cells heterozygous for a knockout mutation in a gene of interest (X) and homozygous for a marker gene (here, black color) are transplanted into the blastocoel cavity of a 4.5-day embryos that are homozygous for an alternative marker (here, white color). The early embryos then are implanted into a peudopregnant female. Some of the resulting progeny are chimeras, indicated by their black and white coats. Chimeric mice then are backcrossed to white mice; black progeny from this mating have ES-derived cells in their germ line. by isolating DNA from a small amount of tail tissue, it is possible to identify black mice heterozygous for the black allele. Intercrossing of these black mice produces individuals homozygous for the disrupted allele, that is, knockout mice. Creation of a Knockout animal General procedure for producing transgenic mice Frequency of random integration of exogenous DNA into the mice genome at nonhomologous sites is very high. Production of transgenic mice is a highly efficient and straightforward process. Cloning in mice. The gene for Human growth hormone was introduced into the genome of the mouse on the right. Expression of the gene resulted in the unusually large size of this mouse  Good bye!! Good luck!!! Hope you learn something useful Hope you do GREAT on the FINAL I’II be around to answer any questions on molecular biology.