13-1 copyright 2005 mcgraw-hill australia pty ltd ppts t/a biology: an australian focus 3e by knox,...

13-1Copyright 2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint

Chapter 13: Genetic engineering and biotechnology


Restriction enzyme mapping

• Restriction endonucleases 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

(cont.)


Fig. 13.1b: Restriction endonucleases


Restriction enzyme mapping (cont.)• 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

(cont.)


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


Fig. 13.2: Electrophoretic separation of fragments


Recombinant DNA molecules

• 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


Fig. 13.3: Ligation of DNA fragments


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

(cont.)


Fig. 13.4: Cloning a human gene (top)


Fig. 13.4: Cloning a human gene (bottom)


DNA vectors (cont.)

• Regardless of their size or origin vector molecules must have the following

– 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


Fig. 13.5a: Plasmid DNA vector


Fig. 13.5b: Selecting cells


Fig. 13.5c: Distinguishing cells


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


Fig. 13.6: Constructing a human genomic library (top)


Fig. 13.6: Constructing a human genomic library (bottom)


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


Fig. 13.7: Constructing a library of cDNA


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


Isolating genes by PCR

• 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


Fig. 13.10: PCR (top)


Fig. 13.10: PCR (bottom)


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


Fig. 13.12a: Sequence determination of a short DNA fragment


Fig. 13.12b: Sequence determination of a short DNA fragment


Fig. 13.12c: Sequence determination of a short DNA fragment


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


Fig. 13.13: Southern (DNA) blotting


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


DNA 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


Fig. 13.18: Find the murderer!


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 (see Fig. 13.20)


Fig. 13.20: Mapping genes to chromosomes by FISH (fluorescence in situ hybridisation)

(a)(b)


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

(cont.)


Fig. 13.23: Animal cloning (top)


Fig. 13.23: Animal cloning (bottom)


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 multipotent stem cells which can be induced to

differentiate in vitro– introduced into patient to replace absent or damaged

cells


Fig. 13.24: Cell therapy

13-1 copyright 2005 mcgraw-hill australia pty ltd ppts t/a biology: an australian focus 3e by knox,...

Documents

molecule dna

dna ligasefig

dna recognition sequences

doublestranded dna

dna sequencescdna libraries

plasmid dna vectorfig

origin vector molecules

different enzymes