DNA-Based Technologies PDF

DNA-based technologies: background, tools, and techniques Marc A. Ilies, Ph. D. Lehninger - Chapter 9 [email protected]; lab 517, office 517A (Tu, Fr 3-5) For questions/comments please use the discussi...

DNA-based technologies: background, tools, and techniques Marc A. Ilies, Ph. D. Lehninger - Chapter 9 [email protected]; lab 517, office 517A (Tu, Fr 3-5) For questions/comments please use the discussion tool in Canvas ©MAIlies2024 1  Genome, genes and the information flow - the genome (total amount of genetic material (DNA/RNA) of a living organism) is the information archive of the cell, encoding all the information needed for cell structure, function and dynamics - DNA molecules are many orders of magnitude longer than the cellular or viral packages that contain them, therefore they are stored condensed (in eukaryotes in chromosomes) - the size of the genome reflects the level of complexity of the organism - gene: DNA piece that encodes the primary sequence of some final product (e.g., a peptide) 2  Recombinant DNA technology "Biotechnology" or "Genetic Engineering": the use of tissue cultures, living cells or cell enzymes to make a defined product - based on the ability to locate, isolate, prepare, and manipulate small segments of DNA from larger chromosomes by processes of DNA cloning. - 1982: Humulin® - human insulin, the first biotech agent FDA-approved DNA cloning involves separating a specific gene or segment from its larger chromosome, attaching it to a small carrier DNA molecule and replicating this many times so that a particular gene or segment is selectively amplified (produce many copies). This process requires the ability to: 1. Cut at precise locations (restriction endonucleases or restriction enzymes); 2. Covalently join two DNA segments (DNA ligase); 3. Join the DNA to be cloned to plasmid or viral DNA (cloning vectors) to produce covalently linked segments; 4. Move recombinant DNA into a host which can provide the enzymes needed for DNA replication. The host may be a bacteria, yeast, mammalian cell or a virus; 5. Select/identify host cells that contain recombinant DNA, separate them and isolate the DNA. 3  Recombinant DNA technology - toolbox: 4  Recombinant DNA technology - major steps: (plasmids: circular DNA molecules that replicate separately from the host chromosome) (via electroporation, lipofection, etc) 5  DNA cleavage and fragmentation - accomplished via bacterial enzymes that have been isolated and used for their unique ability to cleave DNA at particular nucleotide sequences = restriction endonucleases - restriction endonucleases recognise specific palindromic short DNA nucleotide sequences known as restriction sites; they cleave DNA to produce a 3’-OH at one end and a 5’-OH at the other end, resulting in either sticky or blunt ends: - a DNA ligase can join fragments at the sticky ends produced by the same endonucleases to yield recombinant DNA 6 Restriction endonucleases and their specific palindromic sequences 7  Restriction endonucleases and plasmid construction - two different restriction endonucleases are used to isolate a specific sequence: - same restriction endonucleases are used to prepare (cleave) the plasmid: - a ligase will join the insert (linker) to the plasmid: 8  DNA Libraries - a collection of DNA clones, gathered together as a source of DNA for sequencing, gene discovery, or gene function studies; - used to isolate a particular DNA fragment, gene, etc from a larger chromosome; a "library", containing many DNA fragments, is constructed by partial digestion of the DNA with (different) restriction endonucleases; the DNA fragment of interest is identified by taking advantage of its particular sequence: ↓ - we distinguish: - Genomic Library: collection of DNA fragments obtained by digestion of all the DNA of an organism by restriction endonucleases and recombined to a vector. Note: the entire genome contains introns and extrons. - Complementary DNA (c-DNA) Library: a more specialized library which contains only those genes which code for proteins i.e. genes which are transcribed 9 into mRNA.  Making a cDNA library -mRNA of an organism is used to produce complementary DNA by use of reverse transcriptases; - mRNA used as template is destroyed chemically; - A polymerase is used to build the corresponding DNA chain; - the cDNA thus produced is inserted into a vector and cloned forming a cDNA library; - a single strand of cDNA may have a radioactive species attached to it and serve as a probe to find a particular sequence in chromosome. 10  Polymerase Chain Reaction (PCR) - an alternative to older cloning methods that is now extensively used to quickly amplify DNA sequences; requires that sequences (flanking sequences) on either side of the target DNA (the DNA to be amplified) be known - steps: 1. DNA is heated to denature (separate the strands). 2. Synthetic single stranded oligonucleotides complementary to their respective flanking region are added. These act as primers. The separated DNA strands are allowed to cool and the primers are allowed to anneal (base pair). 3. DNA Polymerase and excess deoxyribonucleotides (as NTP) are added. The polymerase adds nucleotides to the 3'-OH end of the primer and the strand grows across the target DNA in the 5'3' direction, making a complementary copy of the target. 4. Reaction is heated again, and steps 2-5 are repeated. Each newly synthesized polynucleotide serves as a template for successive cycles leading to an exponential increase in target DNA, hence the name "chain reaction". - the utility of this process is the result of using a thermo-stable DNA polymerase isolated from thermophilic bacteria. Thus, the reaction consists of repeating cycles of heating and cooling which denature DNA while the polymerase remains stable. Using PCR millions of copies of a gene or a DNA segment can be produced11 very efficiently starting from as low as one molecule of DNA Restriction Fragment Length Polymorphism (RFLP) - there are slight sequence differences ( single base pair changes) that occur from individual to individual once every few hundred base pairs; these changes mean that restriction enzyme sites will vary between individuals so that the sizes of particular restriction fragments will vary; these differences are referred to as RFLPs. Southern Blot - developed by E.M. Southern in 1970’s), is a method to detect RFLPs in DNA: 1. Cleave DNA with restriction endonuclease 2. Separate fragments by gel electrophoresis according to size. 3. Transfer DNA fragments to nitrocellulose nylon membrane. 4. Immerse paper in a solution containing a radioactively labeled DNA probe. 5. Take an X-ray. 6. The fragments which bind the probe will be visualized. - use in “DNA fingerprinting” with forensic uses, paternity tests, prenatal genetic disease diagnosis Principles of the Southern blot technique are used in the Northern blot for RNA and the Western blot for protein analysis 12 Southern Blotting in forensics 13 14  Goals and Objectives Upon completion of this lecture at minimum you should be able to answer the following: ►What is a gene, genome, chromosome, and what is the normal information flow in vivo? ►What is DNA cloning, what it requires, and which are its implications in biotechnology? ►Which are the elements, steps, and processes of DNA recombinant technologies? ►What are restriction endonucleases, how do they act, and which are the results/consequences of their action? ►What is RLFP, where it is used, and what is its significance? ►What is a DNA library, what types of DNA libraries can be distinguished, and how are they generated? ►What is PCR, which are the fundamentals and the steps of the process, and what materials are involved? ►What kind of therapeutic entities can be obtained through recombinant DNA technology? ►What other “omics” do you know? 15  Drugs and diseases ►Diseases: Diabetes ►Drugs: Recombinant human insulin (Humulin), other recombinant products 16