DSCI112 Lecture 17: DNA Tools (Spring 2024) PDF

DSCI112: Molecules, Cells and Organisms Lecture 17: DNA tools [email protected]  Lecture 17: DNA tools Multiple DNA-based tools have been developed that allow us to manipulate DNA in different ways, including amplifying DNA, cutt...

DSCI112: Molecules, Cells and Organisms Lecture 17: DNA tools [email protected]  Lecture 17: DNA tools Multiple DNA-based tools have been developed that allow us to manipulate DNA in different ways, including amplifying DNA, cutting DNA and inserting DNA into different organisms. These tools allow us to perform genetic engineering of genes and organisms. Learning outcomes: Describe what bacterial plasmids are, and how they are used in genetic engineering. Describe how DNA is isolated using restriction enzymes and inserted into a plasmid. Describe how plasmids can be inserted into a bacterium. This lecture is based upon content Describe how genetic engineering within Chapter 20 of Campbell experiments can be analysed. Biology, 12th Edition 2 DNA sequencing and DNA cloning The discovery of the structure of the DNA molecule, and that its two strands are complementary to each other, facilitated the development of DNA sequencing and other techniques for manipulating DNA ‘DNA technology’ is used today in research, medicine and industry  Production of lifesaving drugs, bioremediation, study of diseases The key to these techniques is nucleic acid hybridisation  The base-pairing of one strand of nucleic acid to another (whether DNA or RNA)  The basis of nearly every technique used in genetic engineering 3 Genetic engineering The direct manipulation of genes for practical purposes The process of inserting new genetic information into existing cells in order to modify those cells In fiction: Reality:  Not entirely boring… launched a revolution in the fields of medicine, criminal law, etc. 4 Making Multiple Copies of a Gene or Other DNA Segment When studying a particular gene or group of genes, a molecular biologist faces a challenge:  DNA is very long  A single molecule carries 100s to 1000s of genes  Protein-encoding genes make up only a small proportion of DNA  In order to study a sequence of DNA, one needs to: 1. Isolate the sequence you’re after (separate it from the rest) 2. Make multiple identical copies (provide enough material to work with)  Process called DNA cloning 5 DNA cloning Most methods for cloning piece of DNA in the laboratory share general features  One common approach uses bacteria – often Escherichia coli Bacteria most often contain a single, circular chromosome  Many also contain small circular DNA molecules that replicate independently from the chromosome  ‘Plasmids’ DNA plasmid 6 Plasmids  Double stranded DNA  Circular Chromosome  Non-chromosomal  Can be taken up from the environment  Vary in size (1 billion (109) molecules 30 The Polymerase Chain Reaction PCR is fast and very specific  Only a minuscule amount of DNA need be present in the starting material  This DNA can even be partially degraded, as long as there are a few copies of the complete target sequence Can be used to amplify DNA from a wide variety of sources  A 40,000-year-old frozen woolly mammoth  Hair follicles or tiny amounts of blood, tissue, or semen found at crime scenes  Single embryonic cells for rapid prenatal diagnosis of genetic disorders  Cells infected with viruses that are difficult to detect, such as HIV 31 PCR and cloning Despite its speed, specificity and ability to make large amounts of a target DNA sequence, PCR is no substitution for cloning in cells  Errors can occur, and when they do, they too can be amplified  Lacks the proofreading tools of cells But is still very useful in cloning  Primers can be used to add restriction sites either side of the DNA sequence being targeted! 32 Summary #2 DNA ligases (the same enzymes that join Okazaki fragments together) can be used to ligate digested DNA The Polymerase chain reaction (PCR) is capable of amplifying a target DNA sequence A PCR reaction contains:  Template DNA  DNA polymerase  All four dNTPs  Primers that are complementary to opposite strands at either end of the target DNA sequence The repeated cycles in a PCR reaction are: 1. Denaturation (reaction heated to 95°C) 2. Annealing (reaction is cooled to 50-65°C) 3. Extension (reaction is warmed back up to 72°C) Repeated cycles generated 2n copies of the target DNA (n = # of cycles) Is very fast and specific, and can be used to amplify DNA from many different sources Can be used to add restriction sites to the product to facilitate targeted cloning! 33 Molecular cloning & plasmids 1. Isolate plasmid from bacteria 2. Insert DNA of interest  Produces a recombinant DNA 3. Return plasmid to cell We’re up to this  Recombinant bacterium step now! Many bacteria will naturally take-up DNA from their environment (and those that won't can be made to)  Bacterial ‘Transformation’  But getting the clone you want is not without its problems 34 How to the clone you want? Problem #1: Not all cells will take up plasmids!  Every bacterium in a tube getting a plasmid is not statistically likely… (you might not even have enough plasmid for all of them!)  You need a way to select for only those that do!  Luckily, most plasmids used for genetic engineering have a feature you can use for this  An antibiotic resistance gene  Encodes a gene-product that grants the bacterium resistance to a particular antibiotic  Could be an enzyme that breaks it down, or a membrane pump that extrudes the antibiotic from the cell… 35 Plasmid features Some common cloning plasmid features: A multiple cloning site  Lots of restriction sites all in one place An origin of replication  Got to be able to make more of it Promoters and terminators  Got to make sure the genes work An antibiotic resistance gene  Need to eliminate all the cells that don’t carry the plasmid 36 Antibiotic selection using a resistance marker Basic method: 1. Transform cells with your recombinant plasmid  Foreign DNA ligated into plasmid carrying an antibiotic resistance gene 2. Plate them on media containing that antibiotic 3. Only those bacteria containing the plasmid can produce the product of the antibiotic resistance gene and survive  All surviving cells carry plasmid 37 Antibiotic selection using a resistance marker Allows you to go from looking at this… … to this All cells in a transformation, with no selection Plated on selective media 38 How to the clone you want Plasmid Problem #2: Not all plasmids will be recombinant!  When you digest your DNA Restriction digestion samples, and ligate them together, not all plasmids will pick up the foreign DNA  Some will re-anneal to form the original plasmid, or there may be some in your reaction that was Ligation never digested to begin with…  Both still carry the resistance gene, so how do you tell them apart? Recombinant The original, again? 39 Screening clones for your recombinant construct Sometimes, you clone a gene that has a selectable phenotype  Pretty easy when your gene-of-interest fluoresces under UV light! Other times you need a marker in the plasmid  There are many available,  Most common is the lacZ gene system, and blue-white screening Image credit: Stefan Walkowski - Own work, CC BY-SA 4.0, https://commons.wikimedia.org/ w/index.php?curid=8975334 40 Blue-white screening? Plasmids for Blue-white screening contain a gene called lacZ  This is the E. coli gene that encodes β-galactosidase the enzyme that breaks down lactose LacZ Galactose + Glucose Lactose But how does breaking down lactose help us?  It doesn’t…  But lactose isn’t the only thing it breaks down! 41 Blue-white screening? X-gal is a synthetic product that mimics the structure of lactose  A sugar attached to a dye LacZ  When LacZ cleaves off the sugar, the dye becomes blue, staining the bacteria who have the lacZ gene  How does this help us identify recombinant bacteria?  Look at where we put all our restriction sites  Cloning things in here will mess this gene up! 42 Blue-white screening Non-recombinant plasmids encode LacZ If the lacZ gene has been disrupted by the insertion of foreign DNA however… EcoRI BamHI EcoRI BamHI site site site site P MCS lacZ T P Foreign DNA lacZ Δ T Transcription & Transcription & Translation Translation LacZ  No enzyme  They can produce the enzyme, and if  These cells will not be able to break X-gal is present in the media they can down X-gal in the media, and will remain break it down (and will turn blue) their normal, default ‘white’ colour 43 Blue-white screening Blue colonies are thus non-recombinant  They can survive on the media (because they have the antibiotic resistance gene from the plasmid)  But they don’t have foreign DNA ligated into them  Can still produce functional LacZ White colonies are recombinant  They can survive on the media (because they have the antibiotic resistance gene from the plasmid)  And their copy of the LacZ gene has been disrupted by the insertion of the foreign DNA  Know precisely which colonies to pick up for later use 44 Summary #3 Process of making a recombinant plasmid:  Digest (cut) the plasmid & the foreign genome with a restriction enzyme with a restriction enzyme  Digest (cut) the foreign genome with a restriction enzyme, to cut out foreign gene of interest  Foreign gene and plasmid will H-bond through complementary (sticky) ends  Foreign gene can then be ‘ligated’ into plasmid with DNA ligase, which will covalently bond the sugar- phosphate backbone Transforming a recombined plasmid (plasmid with a foreign gene ligated into it) into bacteria:  Recombined and empty plasmids in the sample will be transformed into bacteria  All plasmids will have a gene for antibiotic resistance (whether they have the foreign gene or not) ▪ Non-transformed bacteria with no plasmids inside will die Some plasmids have a lacZ gene, which codes an enzyme which will cut X-gal to make a blue pigment  If the foreign gene is successfully ligated into the plasmid, the plasmid will NOT make the enzyme: ▪ White colonies – white indicates the gene of interest has cloned into the plasmid ▪ Blue colonies – blue indicates the lacZ gene is intact, so the gene of interest has not cloned into the plasmid 45