DNA Technology PDF

Chapter 19 DNA Technology Lecture Presentations by Nicole Tunbridge and © 2021 Pearson Education Ltd. Kathleen Fitzpatrick Figure 19.1b...

Chapter 19 DNA Technology Lecture Presentations by Nicole Tunbridge and © 2021 Pearson Education Ltd. Kathleen Fitzpatrick Figure 19.1b © 2021 Pearson Education Ltd. CONCEPT 19.1: DNA sequencing and DNA cloning are valuable tools for genetic engineering and biological inquiry The main technologies for sequencing and manipulating DNA are called DNA technology The complementarity of the two DNA strands is the basis for nucleic acid hybridization, the base pairing of one strand of nucleic acid to the complementary sequence on another strand Genetic engineering is the direct manipulation of genes for practical purposes © 2021 Pearson Education Ltd. The Human Genome Project The first full human genome sequence was completed in 2003 It took 13 years to be completed It cost $1billion The time and cost of genome sequencing have been greatly reduced by improved methods © 2021 Pearson Education Ltd. DNA Sequencing A gene’s complete nucleotide sequence can be determined using a process called DNA sequencing The first automated procedure was based on a technique called dideoxy or chain termination sequencing, developed by Frederick Sanger (Sanger Sequencing) In the first decade of this century, “next-generation sequencing” (NGS) techniques have been developed that are rapid and inexpensive © 2021 Pearson Education Ltd. Figure 19.2 Next-generation DNA sequencing machines © 2021 Pearson Education Ltd. Video: Sanger Method of DNA Sequencing © 2021 Pearson Education Ltd.  In sequencing by synthesis (NGS), many DNA fragments are copied to produce an enormous number of identical fragments A single strand of each fragment is immobilized and the complementary strand synthesized one nucleotide at a time Thousands or hundreds of thousands of fragments about 300 nucleotides long can be sequenced in parallel This is an example of “high-throughput” technology © 2021 Pearson Education Ltd. Figure 19.3 Sequencing by Synthesis: Next-Generation Sequencing © 2021 Pearson Education Ltd.  “Third-generation sequencing” techniques are even faster and less expensive In these methods, a single long DNA molecule is sequenced as it moves through a very small pore (nanopore) in a membrane In one approach, each base is identified by the way it interrupts an electric current as it passes through the pore Associated software allows identification and analysis of the DNA sequence © 2021 Pearson Education Ltd. Figure 19.1a A DNA strand is passed through a pore in a membrane. The resulting changes in an electrical current are used to determine the nucleotide sequence. © 2021 Pearson Education Ltd. Making Multiple Copies of a Gene or Other DNA Segment To work directly with specific genes, scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning Plasmids are small, circular DNA molecules that replicate separately from the bacterial chromosome Researchers can insert DNA into a plasmid to produce a recombinant DNA molecule, which contains DNA from two different sources © 2021 Pearson Education Ltd. Figure 19.4 © 2021 Pearson Education Ltd. Animation: Recombinant DNA © 2021 Pearson Education Ltd.  Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA This production of multiple copies of a single gene is a type of DNA cloning called gene cloning A plasmid used to clone a foreign gene is called a cloning vector © 2021 Pearson Education Ltd.  Bacterial plasmids are widely used as cloning vectors because they are: – readily obtained – easily manipulated – easily introduced into bacterial cells – rapidly multiplied once in the bacteria Gene cloning is useful for amplifying genes to produce a protein product for research, medical, or other purposes © 2021 Pearson Education Ltd. Using Restriction Enzymes to Make a Recombinant DNA Plasmid Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites A restriction enzyme usually makes many cuts in a long DNA molecule, yielding restriction fragments The most useful restriction enzymes cut DNA in a staggered way, producing fragments with at least one single-stranded end called a sticky end © 2021 Pearson Education Ltd.  Sticky ends can bond with complementary sticky ends of other fragments DNA ligase is an enzyme that seals the bonds between restriction fragments This allows researchers to join two DNA fragments from different sources © 2021 Pearson Education Ltd. Animation: Restriction Enzymes © 2021 Pearson Education Ltd. Figure 19.5 Using a restriction enzyme and DNA ligase to make a recombinant DNA plasmid © 2021 Pearson Education Ltd.  To check the recombinant plasmid, researchers might cut the products again using the same restriction enzyme To separate and visualize the fragments produced, gel electrophoresis is carried out This technique uses a gel made of a polymer that has microscopic holes of different sizes, through which shorter fragments can travel faster © 2021 Pearson Education Ltd. Figure 19.6 © 2021 Pearson Education Ltd. Animation: Gel Electrophoresis of DNA https://www.youtube.com/watch?v=MhJT9yjnl88 © 2021 Pearson Education Ltd. Amplifying DNA: The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA A three-step cycle—heating (denaturing), cooling (annealing), and extension—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules The process uses primers, short single-stranded DNA molecules complementary to sequences to either side of the target sequence © 2021 Pearson Education Ltd.  The key to PCR is an unusual, heat-stable DNA polymerase called Taq polymerase Other polymerases may be used as well; some are more accurate and stable than Taq, such as Pfu polymerase The primers used are specific for the sequence to be amplified PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells © 2021 Pearson Education Ltd. Figure 19.7 © 2021 Pearson Education Ltd.  PCR is used to produce the specific DNA fragment for cloning PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors The resulting clones are sequenced and error-free inserts selected © 2021 Pearson Education Ltd.