Transcript 14-31 - McGraw Hill Higher Education

Chapter 14: Genetic engineering and
biotechnology
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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
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Fig. 14.1: Restriction endonucleases
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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
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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
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Fig. 14.2: Electrophoretic separation of fragments
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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
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Fig. 14.4: Ligation of DNA fragments
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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
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Fig. 14.5: Cloning a gene (top)
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Fig. 14.5: Cloning a gene (bottom)
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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
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Fig. 14.6a: Plasmid DNA vector
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Fig. 14.6b: Selecting cells
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Fig. 14.6c: Plating transformed cells
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Fig. 14.6d: Distinguishing cells
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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
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Fig. 14.7 (top): Constructing a genomic library
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Fig. 14.7 (bottom): Constructing a genomic library
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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
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Fig. 14.8: Constructing a library of cDNA
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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
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Fig. 14.9: Colony hybridisation method
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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
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Fig. 14.10 (top): Polymerase chain reaction
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Fig. 14.10 (bottom): Polymerase chain reaction
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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
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Fig. 14.12: Southern (DNA) blotting
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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
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Fig. 14.11: Automated enzymatic DNA sequencing
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Question:
Now that you have a DNA sequence, what can you
do with this information?
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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
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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
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Fig. 14.18: Find the murderer!
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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
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Fig. 14.19:
Human X chromosome
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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
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Fig. 14.22 (top): Animal cloning
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Fig. 14.22 (bottom): Animal cloning
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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
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Cell therapy using embryonic stem cells
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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
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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
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14-43