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Chapter 14: Genetic engineering and biotechnology 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 14-1 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 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 14-2 Fig. 14.1: Restriction endonucleases 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 14-3 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 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 14-4 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 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 14-5 Fig. 14.2: Electrophoretic separation of fragments 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 14-6 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 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 14-7 Fig. 14.4: Ligation of DNA fragments 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 14-8 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 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 14-9 Fig. 14.5: Cloning a gene (top) 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 14-10 Fig. 14.5: Cloning a gene (bottom) 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 14-11 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 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 14-12 Fig. 14.6a: Plasmid DNA vector 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 14-13 Fig. 14.6b: Selecting 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 14-14 Fig. 14.6c: Plating transformed 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 14-15 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 14-16 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 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 Fig. 14.7 (top): Constructing a genomic library 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 14-18 Fig. 14.7 (bottom): Constructing a genomic library 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 14-19 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 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 14-20 Fig. 14.8: Constructing a library of cDNA 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 14-21 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 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 14-22 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 14-23 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 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 Fig. 14.10 (top): Polymerase chain reaction 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 14-25 Fig. 14.10 (bottom): Polymerase chain reaction 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 14-26 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 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 14-27 Fig. 14.12: Southern (DNA) blotting 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 14-28 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 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 14-29 Fig. 14.11: Automated enzymatic DNA sequencing 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 14-30 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 14-31 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 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 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 repeatsequence 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 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 14-33 Fig. 14.18: Find the murderer! 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 14-34 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 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 14-35 Fig. 14.19: 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 14-36 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 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 Fig. 14.22 (top): Animal cloning 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 14-38 Fig. 14.22 (bottom): Animal cloning 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 14-39 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 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 14-40 Cell therapy using embryonic stem 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 14-41 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 14-42 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 14-43
