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Chapter 17

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Wilfrid Laurier University
Robert Boulianne

Chapter 17 – Recombinant DNA Technology and Gene Cloning - Recombinant DNA - A DNA molecule formed by joining together DNA from different biological sources - Cloning can produce millions of copies of this recombinant DNA molecule - The sequence can also be manipulated – Changing the sequence - Produce the protein and new proteins - Steps in Cloning – 1. Purify DNA 2. Cut DNA (with restriction enzymes) 3. Join DNA to other DNAs that serve as vectors or carrier molecules (eg. Plasmids) 4. Recombinant DNA transferred to a host cell 5. Allow host cell to grow and divide thereby replication the recombinant DNA molecule as well 6. Purify cloned DNA from host cells for analysis 7. Cloned DNA may also be transcribed to produce the gene product for research or commercial purposes -Restriction enzymes – are proteins that are used to generate specific DNA fragment and these enzymes recognize and cut DNA molecules at specific nucleotide sequences called the recognition site/sequence into restriction fragments leaving complementary tails on both sides of the fragment Eg. Hind11 and BamHI and Sau3AI enzyme make offset staggered cuts in the DNA strands, fragments with short, single-stranded tails that anneal (stick together) with different DNA source AluI cut both strands at the same nucleotide pair producing blunt ends - A DNA sequence is palindromic when the complementary strand of a DNA sequence is the same from it being 5’-3’ both ways Constructing Recombinant DNA molecules -DNA from different sources is cleaved with the same enzyme (EcoRI) and mixed to allow annealing. -Annealing allows recombinant DNA molecules to form by complementary base pairing - The two strands are not covalently bonded at the where there is a gap - DNA ligase seals the gaps, covalently bonding the two strands together - Fragments of DNA can’t be introduced into a host cell directly - They don’t have the necessary sequences for replication, copy number and selectable markers - Therefore Vectors (Plasmids) are used - Vectors and its inserted DNA fragment should be easy to recover from host cell - Vectors carry out 3 important functions: - Replicate independently along with DNA fragment it carries once in host cell (it has an origin of replication) - Contain several restriction sites for different insertions - Carries a selectable marker (usually antibiotic resistance) which identifies host cells that contain vector and maintains the vector in the host cell - Plasmids are naturally occurring, extrachromosomal and self-replicating dsDNA molecules found in certain bacteria - Over the years plasmids have been extensively modified to serve as cloning vectors for a variety of applications - Vectors containing an inserted fragment are called recombinant vectors - pUC 18 Plasmid is small, 2686 bp - Origin of replication, can produce up to 500 copies per cell - Large number of restriction enzyme sites for cloning, especially in polylinker site - Selectable marker for ampicillin resistance (β-lactamase) -Polylinker in the lacZ gene for blue-white colour selection - DNA inserted into the polylinker region disrupts the lacZ gene resulting in white colonies that allow direct identification of bacterial colonies carrying cloned DNA inserts - Active lacZ gene product cleaves Xgal into blue product - Expression of lacZ causes bacterial host cells carrying pUC18 to produce blue colonies when grown on medium containing a compound known as Xgal - Inactive lacZ gene product can’t cleave Xgal, so cells are white - Cloning into Other Microorganisms - Yeast Artificial Chromosomes (YACs) - Made to clone large fragments (up to a million bp) - Useful in human genome project - Has telomeres at each end, an orgin of replication, and a centromere - Cloning DNA in Host Cells - Plasmid is removed from bacterial cell and cut with a restriction enzyme - DNA to be cloned is cut with the same restriction enzyme - The two DNAs are ligated to form a recombinant molecule by treating it with DNA ligase - Introduced into host cell to be replicated - Cells carrying recombinant plasmids can be selected or screened by plating on medium containing antibiotics or color indicators such as Xgal - For Expression of Eukaryotic Genes - Yeast is the host of choice (YACs) - Eukaryotic proteins may need to be modified to have optimal activity (eg. Through the ER or golgi) which bacteria lack these organelles - Some example of recombinant proteins synthesized in Yeast cells are Hepatitis B virus surface protein, Malaria parasite protein, Epidermal growth factor….etc - The Polymerase Chain Reaction (PCR) - In 1986 a technique was developed that accelerated recombinant DNA methodology - Kary Mullis won 1993 Nobel Prize in Chemistry - PCR is a rapid method of copying DNA in vitro where there is no need for host cell to amplify DNA - Method of choice in most cloning protocols because it Produces billions of copies of a specific DNA sequence in a matter of hours - Steps in PCR - 3Steps: Denaturation at 95°C which separates dsDNAs into ssDNA - Annealing at 50°-70°C - ssDNA primers base pair with specific sequences on target DNA (annealing) - Temperature varies and depends on Tm of primer - Extension at 72°C - Optimal temperature for TAQ polymerase activity - Extends primers from 5’ to 3’ - These three steps comprise 1 cycle. A typical PCR protocol calls for 25 to 30 cycles - Each cycle takes 2 to 5 mins - The amount of target DNA will double with each cycle - Important enzyme used is TAQ Polymerase - Isolated DNA polymerase from Thermophilus aquaticus, a bacterial strain isolated from hot springs - Its enzymes are stable at high temperatures, so TAQ won’t be denatured with repeating denaturing temperatures of 95°C during PCR cycling - cDNA Library - Contains DNA copied from mRNA in a cell population at a given time - Remember that not all genes are expressed at same time, so a cDNA library will only have a subset of the genes from the entire genome - Eg. We could create a cDNA library for liver cells and another cDNA library for skin cells (some mRNAs would be present in both, while some others in only skin and others only in liver) - cDNA stands for complementary DNA - We can only clone dsDNA into vectors for cloning into a library - Challenge is to make a dsDNA copy of mRNA - To make the cDNA library the enzyme Reverse Transcriptase will be used to make DNA copy of mRNA - cDNA library is prepared by first isolating mRNAs from a population of cells and using the mRNAs as templates for the synthesis of double-stranded cDNA molecules - then inserting them into vectors and cloned to produce a library that is a snapshot of genes that were transcriptionally active at a given time - Steps into making a cDNA library - mix the mRNAs that have Poly-A-tail with oligo-dT primer (A strand of nucleotides with all TTTT) - so the oligo-dT primer will anneal with the Poly-A-tail, forming a partially double-stranded product - Reverse Transcriptase is added to extend the primer and synthesize a mRNA – DNA double stranded hybrid molecule - Action of the enzyme RNase H creates nicks in the RNA strand by partially digesting the RNA ( Eats some of the RNA strand in intervals, leaving bits) - is added and the remaining RNA fragments serves as primers ( This situation is similar to synthesis of the lagging strand of DNA prokaryotes) - DNA polymerase I removes RNA primers and synthesize a second DNA strand - DNA ligase is added to seal gaps - cDNA can be inserted into a vector by attaching linker sequences to the blunt ends of the cDNA - Linker sequences – short double stranded oligonucleotides containing a restriction enzyme recognition sequence (eg. EcoRI) - After the attachment, the linkers are cut with EcoRI, producing fragments with single stranded tails - These fragments are then ligated to vectors treated with the same enzyme - Retrieval of Specific Clones from Libraries - A probe is used to screen a library to recover a specific gene - The probe can be
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