14-1 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Chapter 14: Genetic engineering and biotechnology

14-2 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Cutting and joining DNA Restriction endonucleases (aka. restriction enzymes) cut double-stranded DNA at defined sequences Each restriction enzyme cuts a particular palindromic sequence The enzymes have been isolated from bacteria which use them to inactivate foreign DNA Identical DNA molecules will be cut into fragments of the same length based on the position of the endonuclease recognition sites on the molecule

14-3 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Fig. 14.1: Restriction endonucleases

14-4 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Restriction enzyme mapping Cutting identical molecules with different enzymes produces a different pattern of fragments The patterns will overlap—cutting with two enzymes together produces a greater number of smaller fragments which are equivalent in total length to either enzyme alone This allows the relative positions of the DNA recognition sequences to be mapped

14-5 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Restriction enzyme mapping (cont.) Fragments are separated by size using gel electrophoresis The electric current causes fragment migration through the gel, with small fragments moving faster than large fragments

14-6 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Fig. 14.2: Electrophoretic separation of fragments

14-7 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Recombinant DNA technology Restriction enzymes cut at defined sites regardless of the origin of the molecule DNA from different sources can be joined to form a recombinant molecule as long as the same restriction enzyme was used to cut each molecule Some enzymes produce staggered cuts in which short single-stranded regions protrude The molecules adhere at these sites and are ligated together by DNA ligase

14-8 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Fig. 14.4: Ligation of DNA fragments

14-9 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University DNA vectors Production of multiple copies of the DNA fragment requires ligation into a self-replicating vector molecule –plasmids –bacteriophage –cosmids –YACs (yeast artificial chromosomes) and –BACs (bacterial artificial chromosomes) Replication of the recombinant vector occurs in the appropriate bacterial or yeast host

14-10 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Fig. 14.5: Cloning a gene (top)

14-11 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Fig. 14.5: Cloning a gene (bottom)

14-12 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University DNA vectors (cont.) Regardless of their size or origin, vector molecules must have: –an origin of replication –at least one unique restriction site for insertion of DNA fragment –a gene for an inducible character, such as antibiotic resistance, to ensure efficient replication in the host organism –a means of distinguishing between vector alone and recombinant vector molecules

14-13 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Fig. 14.6a: Plasmid DNA vector

14-14 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Fig. 14.6b: Selecting cells

14-15 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Fig. 14.6c: Plating transformed cells

Fig. 14.6d: Distinguishing cells Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University

14-17 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Genomic DNA libraries Entire genomes are fragmented and ligated into a vector Millions of resulting colonies or plaques are produced, each one of which contains a piece of the genome If the library is large enough, each fragment of genome should be present at least once

14-18 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Fig (top): Constructing a genomic library

14-19 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Fig (bottom): Constructing a genomic library

14-20 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University cDNA libraries Genomic DNA libraries contain all DNA sequences cDNA libraries contain only those coding sequences present in transcribed genes mRNA molecules are copied by reverse transcriptase into complementary cDNA cDNA molecules are ligated into vectors and a library constructed Each clone is derived from a gene being expressed at the time of the mRNA isolation

14-21 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Fig. 14.8: Constructing a library of cDNA

14-22 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Identifying cloned sequences Hybridisation –colonies or plaques grown on plates –recombinant DNA in the colonies is denatured –a replica of the plate is made on a membrane filter and the adherent cells lysed to reveal their DNA –a labelled, single-stranded probe to the gene of interest is hybridised to complementary sequences on the membrane –the original colony or plaque can be recovered from the plate and used in further analysis

Fig. 14.9: Colony hybridisation method Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University

14-24 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Isolating genes by PCR amplification Polymerase chain reaction (PCR) allows the amplification of specific sequences without the need for cells –amplification is selective and repeated, using heat-stable DNA polymerase and deoxynucleotide triphosphates –specificity is determined by the use of oligonucleotide primers to known sequences flanking the fragment of interest –each cycle of annealing and extension doubles the fragment copy number

14-25 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Fig (top): Polymerase chain reaction

14-26 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Fig (bottom): Polymerase chain reaction

14-27 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University DNA (and RNA) blotting Called Southern blotting after its inventor Edwin Southern –DNA isolated and cut into different-sized fragments –fragments separated physically by size using gel electrophoresis –separated fragments are denatured and transferred to a membrane filter –radiolabelled single-strand probe is bound to the fragment of interest, making it visible A similar technique is used to identify mRNA molecules

14-28 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Fig : Southern (DNA) blotting

14-29 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Nucleotide sequence analysis The base sequence of DNA can be determined in vitro by DNA synthesis and electrophoresis –each synthesis reaction contains normal deoxynucleoside triphosphates and a chain-terminating dideoxynucleoside triphosphate (ddNTP) –four reactions are employed, each containing a different ddNTP to stop the reaction –a series of fragments is generated with different lengths but each terminating in the same nucleotide (the ddNTP) –each reaction is labelled with a different colour and the sequence read as a series of fluorescent bands

14-30 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Fig : Automated enzymatic DNA sequencing

Question: Now that you have a DNA sequence, what can you do with this information? Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University

14-32 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Analysing genetic variation Base changes in a gene result in restriction fragment length polymorphisms (RFLPs) The consistent presence of a particular RFLP in people with the disease being investigated is strong evidence of the mutation causing the disease—also permits localisation of the gene in which the mutation has occurred RFLPs can be distinguished by Southern hybridisation or by PCR

14-33 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University DNA technology in forensic science Developed as a way of defining specific differences in DNA sequences between people –differences must be extensive and detailed enough to minimise risk of accidental identity –gene sequences are not used for this –microsatellites and minisatellites: regions of repeat- sequence DNA, where short sequences (2–5 nucleotides) may be repeated many times –VNTRs (variable number tandem repeats) are similar. They vary in number between individuals, so looking at several VNTRs at once provides a unique ‘fingerprint’ of sequence lengths for that person

14-34 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Fig : Find the murderer!

14-35 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Mapping genes Classical gene linkage analysis has limitations, especially in mammals DNA sequence polymorphisms can be used as landmarks to detect recombination in offspring of heterozygous parents Association of linkage markers with disease alleles is important in the location and isolation of the disease gene The physical location on a chromosome of a gene can be found using a labelled probe from a cloned sequence

Fig : Human X chromosome Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University

14-37 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Biotechnology Recombinant protein production –gene products such as drugs, hormones and enzymes can be produced in large quantities in cell culture systems Modifying agricultural organisms –inserting genes for improved yield or pest resistance into plants –cloning domestic animals chosen for their superior qualities

14-38 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Fig (top): Animal cloning

14-39 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Fig (bottom): Animal cloning

14-40 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Biotechnology (cont.) Gene therapy –the introduction of a modified gene into the cells of a patient suffering a genetic disease to correct the abnormality –still experimental –problems associated with directing the vector to the target cells and maintaining expression Cell therapy –the use of stem cells, which can be induced to differentiate in vitro –introduced into patient to replace absent or damaged cells

14-41 Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University Cell therapy using embryonic stem cells

Summary Recombinant DNA techniques isolate genes or small segments of DNA from chromosomes Specific cuts in DNA molecules can be made by specialised enzymes Fragments can be separated and sized by using gel electrophoresis DNA fragments can be joined (ligated) to form recombinant DNA molecules PCR technique offers a rapid means of obtaining sizable quantities of genes and DNA fragments from small amounts of DNA Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University

Summary (cont.) Cloned DNA molecules can be analysed by using restriction enzymes and direct sequencing DNA technology enables us to identify genetic variation in terms of changes in base sequences Recombinant DNA technology can be used to change the genetic make-up of organisms by genetic modification Controlled growth and differentiation of stem cells may in the future offer therapy for disease or injury Copyright  2010 McGraw-Hill Australia Pty Ltd PowerPoint slides to accompany Biology: An Australian focus 4e by Knox, Ladiges, Evans and Saint Slides prepared by Karen Burke da Silva, Flinders University