DNA Technology and Genomics
Why?
• Understand the way in which plants and
animals, including humans, develop,
function and evolve
• Investigate the molecular basis of disease
• Develop products for medicine and crops
for agriculture
• Solve crimes and paternity disputes
• Investigate endangered species for
conservation management
Studying DNA
• A number of methods have been developed that
can be used to identify the DNA (genetic) profile
of an individual
• These methods can also be employed to
measure genetic differences between individuals
in a population
• Techniques for working with DNA can be broken
down into four major categories:
–
–
–
–
Copying DNA
Cutting and pasting DNA
Measuring DNA length
Probing DNA
Extracting DNA
• Break open (lyse) the cells or virus containing the DNA of interest.
This is often done by sonicating or bead beating the sample.
Vortexing with phenol (sometimes heated) is often effective for
breaking down protienacious cellular walls or viral capsids. The
addition of a detergent such as SDS is often necessary to remove
lipid membranes.
• DNA associated proteins, as well as other cellular proteins, may be
degraded with the addition of a protease. Precipitation of the protein
is aided by the addition of a salt such as ammonium or sodium
acetate. When the sample is vortexed with phenol-chloroform and
centrifuged the proteins will remain in the organic phase and can be
drawn off carefully. The DNA will be found at the interface between
the two phases.
• DNA is the precipitated by mixing with cold ethanol or isopropanol
and then centrifuging. The DNA is insoluble in the alcohol and will
come out of solution, and the alcohol serves as a wash to remove
the salt previously added.
• Wash the resultant DNA pellet with cold alcohol again and
centrifuge for retrieval of the pellet.
• After pouring the alcohol off the pellet and drying, the DNA can be
re-suspended in a buffer such as Tris or TE.
Why we need so many copies
• Biologists needed to find a way to read DNA
codes.
• How do you read base pairs that are angstroms
in size?
– It is not possible to directly look at it due to DNA’s
small size.
– Need to use chemical techniques to detect what
you are looking for.
– To read something so small, you need a lot of it,
so that you can actually detect the chemistry.
• Need a way to make many copies of the base
pairs, and a method for reading the pairs.
Polymerase Chain Reaction
(PCR)
• Polymerase Chain Reaction (PCR)
– Used to massively replicate DNA sequences.
– Exploits enzymes and process of replication
that normally occurs in cells.
• How it works:
– Separate the two strands with low heat
– Add some base pairs, primer sequences, and
DNA Polymerase
• Creates double stranded DNA from a single
strand.
• Primer sequences create a seed from which
double stranded DNA grows.
– Now you have two copies.
– Repeat. Amount of DNA grows exponentially.
• 1→2→4→8→16→32→64→128→256…
Polymerase Chain Reaction
• Problem: Modern
instrumentation cannot
easily detect single
molecules of DNA,
making amplification a
prerequisite for further
analysis
• Solution: PCR doubles
the number of DNA
fragments at every
iteration
1…
2…
4…
8…
Polymerase Chain Reaction
• Polymerase Chain Reaction (PCR) is broken down into three
separate steps which are repeated until enough DNA is obtained
(usually between 25 and 40 cycles)
• Step 1 – Denaturation
– Temperature is raised (94oC) in order to separate dsDNA into single
strands
• Step 2 – Annealing
– Temperature decreased (50-60oC) in order for primers to anneal and
provide a starting point for DNA polymerase
• Step 3 – Extension
– Temperature increased (72oC) which allows Taq polymerase to extend
DNA
– Taq polymerase is a heat resistant DNA polymerase isolated from the
bacterium Thermus aquaticus which is found in hot springs and
hydrothermal vents
Denaturation
Raise temperature to 94oC
to separate the duplex form
of DNA into single strands
Design primers
• To perform PCR, a 10-20bp sequence on either
side of the sequence to be amplified must be
known because DNA polymerase requires a primer
to synthesize a new strand of DNA
Annealing
• Anneal primers at 50-65oC
Annealing
• Anneal primers at 50-65oC
Extension
• Extend primers: raise temp to 72oC, allowing Taq
pol to attach at each priming site and extend a
new DNA strand
Extension
• Extend primers: raise temp to 72oC, allowing Taq
pol to attach at each priming site and extend a
new DNA strand
Repeat
• Repeat the Denature, Anneal, Extension
steps at their respective temperatures…
Polymerase Chain Reaction
RT-PCR: Variation of PCR
• PCR reaction amplifies DNA from a single copy in the absence of
cells
• RT-PCR is a variant of PCR in which DNA is first reverse
transcribed (copied in reverse) from RNA extracted from cells prior
to being amplified by PCR as per usual protocol
• Reverse transcription process involves the use of a reverse
transcriptase enzyme and various other reagents including either a
primer for a specific gene or an oligo-dT primer (string of Ts that will
bind to poly-A tail of RNA.
• Enzyme, reagent mix, primer and RNA are usually incubated at
37oC for 1 hour before the RT enzyme is inactivated at 72oC for
approximately 15 minutes. The resulting cDNA is then used as the
template for a PCR reaction.
RT-PCR
PCR begins
here
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•
•
Cloning DNA to achieve copies
Use restriction enzymes and DNA ligase
to insert the fragment of interest into the
genome of another organism (e.g.
bacteria) in order for it to multiply.
The resulting DNA is referred to as
recombinant DNA as the genes from two
different organisms are combined.
Once you have a large quantity of
bacteria, you will be able to isolate a large
quantity of the gene of interest
Vector DNA
Restriction Enzymes
• Discovered in the early 1970’s
– Used as a defense mechanism by bacteria to
break down the DNA of attacking viruses.
– They cut the DNA into small fragments.
• Can also be used to cut the DNA of organisms.
– This allows the DNA sequence to be in a
more manageable bite-size pieces.
• It is then possible using standard purification
techniques to single out certain fragments and
duplicate them to macroscopic quantities.
Restriction Enzymes
Definition:
A restriction enzyme is a bacterial enzyme that
recognises a short sequence of bases in a DNA
molecule and cuts the DNA at this recognition site.
• The position where a cutting enzyme can snip is its
recognition sequence and is where a particular order of
nucleotides occurs.
• Some restriction enzymes cut the two strands of a DNA
molecule at points directly opposite each other to
produce cut ends that are ‘blunt’.
• Other cutting enzymes cut one strand at one point, but
cut the second strand at a point that is not directly
opposite.
• The overhanging cut ends made by these cutting
enzymes are called ‘sticky’. These sticky ends are
complementary.
Blunt and Sticky Ends
Pasting DNA
• Once separated, DNA
fragments from different
sources can be joined
(ligated) together.
• Sticky ended fragments will
initially join by hybridization or
complementary base pairing.
• Bonds within the single
strands of DNA are then
repaired by DNA ligase (this
is similar to the action of DNA
ligase in linking of Ozaki
fragments on the lagging
strand during DNA
replication)
Gel Electrophoresis
• A copolymer of mannose and galactose, agaraose,
when melted and recooled, forms a gel with pores sizes
dependent upon the concentration of agarose.
• The phosphate backbone of DNA is highly negatively
charged, therefore DNA will migrate in an electric field.
Gel Electrophoresis
• The size of DNA fragments can then be determined by comparing
their migration in the gel to known size standards
• Ethidium bromide or other dyes that bind to DNA are added prior to
electrophoresis in order to visualize migration of DNA
• Ethidium bromide flouresces bright orange when exposed to UV
light
Reading DNA – DNA Sequencing
•
DNA sequencing reactions are just like the PCR reactions for replicating
DNA with the exception that the reactions are run in the presence of a
dideoxynucleotide.
•
Dideoxyribonucleotides are the same as nucleotides, with one exception.
They do not have 3' hydroxyl group, so once a dideoxynucleotide is added
to the end of a DNA strand, there's no way to continue elongating it.
•
Sequencing reactions are set up in groups of four, e.g. one containing
dideoxy-A, one containing dideoxy-C, one containing dideoxy-G and one
containing dideoxy-T. Each reaction tube contains a mix of normal
nucleotides (A,C,G, T) and a small amount of the particular
dideoxynucleotide.
•
Taking dideoxy-C as an example, replication of DNA will occur as per a
PCR reaction. MOST of the time when a ‘C' is required to make the new
strand, the enzyme will get a good one and there's no problem. MOST of
the time after adding a C, the enzyme will go ahead and add more
nucleotides. However, 5% of the time, the enzyme will get a dideoxy-C, and
that strand can never again be elongated. It eventually breaks away from
the enzyme, a dead end product.
Reading DNA – DNA Sequencing
•
•
Sooner or later ALL of the copies will get terminated by a T, but each time the
enzyme makes a new strand, the place it gets stopped will be random. In
millions of starts, there will be strands stopping at every possible T along the
way.
ALL of the strands we make started at one exact position. ALL of them end
with a T. There are billions of them ... many millions at each possible T
position. To find out where all the T's are in our newly synthesized strand, all
we have to do is find out the sizes of all the terminated products!
Reading DNA – DNA Sequencing
•
•
•
Gel electrophoresis can be used to separate the fragments
by size and measure them.
The dideoxynucleotides present in the fragments have been
labelled with a radioisotope or a flourescent dye. In the case
of the latter, these can be read by a laser and the
information feed back to computer.
Following electrophoresis and visualization of fragments we
can determine the sequence. Smallest fragments are at the
bottom, largest at the top. The positions and spacing shows
the relative sizes. At the bottom are the smallest fragments
that have been terminated by dideoxynucleotides.
Assembling Genomes
• Based on sequencing data, we can take fragments and
put them back together.
Not as easy as it sounds!!!!!
• SCS Problem (Shortest Common Superstring)
– Some of the fragments will overlap
– We try to fit overlapping sequences together to get the shortest
possible sequence that includes all fragment sequences
– Problems that may arise during this process:
• DNA fragments contain sequencing errors
• There are two complements of DNA – we need to take into account
both directions of DNA
• The repeat problem - 50% of human DNA is just repeats. If you
have repeating DNA, how do you know where it goes?
Analyzing a Genome
• How to analyze a genome in four easy steps.
– Cut it
• Use enzymes to cut the DNA in to small fragments.
– Copy it
• Copy it many times to make it easier to see and detect.
– Read it
• Use special chemical techniques to read the small fragments.
– Assemble it
• Take all the fragments and put them back together. This is
hard!!!
• Bioinformatics takes over
– What can we learn from the sequenced DNA.
– Compare interspecies and intraspecies.
Nucleotide Hybridization
• Single-stranded DNA or RNA will naturally bind to complementary
strands.
• Hybridization is used to locate genes, regulate gene expression, and
determine the degree of similarity between DNA from different
sources.
• Hybridization is also referred to as annealing or renaturation.
• Hybridization uses oligonucleotides to find complementary DNA or
RNA seqments. Oligonucleotides are single-stranded DNA molecules
of 20-30 nucleotides in length.
• Oligonucleotides are made with DNA synthesizers and tagged with a
radioactive isotope or fluorescent dye
• Molecular techniques based on hybridization include Southern blotting,
Northern blotting and microarrays.
Create a Hybridization
Reaction
1.
2.
Hybridization is binding two genetic
sequences. The binding occurs
because of the hydrogen bonds [pink]
between base pairs.
When using hybridization, DNA must
first be denatured, usually by using
use heat or chemical.
T
C
A
G
T
TAGGC T G
T
C
G
CT
A
T
ATCCGACAATGACGCC
Create a Hybridization Reaction Cont.
3.
4.
Once DNA has been denatured, a
single-stranded radioactive probe [light
blue] can be used to see if the
denatured DNA contains a sequence
complementary to probe.
Sequences of varying homology stick
to the DNA even if the fit is poor.
ACTGC
ACTGC
ATCCGACAATGACGCC
Great Homology
ACTGC
ATCCGACAATGACGCC
Less Homology
ATTCC
ATCCGACAATGACGCC
ACCCC
ATCCGACAATGACGCC
Low Homology
Southern Blotting
•
•
•
•
•
•
Cut total genomic DNA with restriction
enzymes and separate by electrophoresis
Blot the fragments onto nitrocellulose filter
paper (the Southern blot)
Probe the blot for a particular DNA region
of interest using a specific labelled
oligonucleotide.
Wash blot to remove oligonucleotide that
has not bound.
Identify gene or region of interest by
visualizing regions where probe has
hybridised with DNA on Southern blot.
Northern blotting follows a similar process
to Southern blotting except that RNA is
run on the initial gel and the
oligonucleotide probes are used to detect
expression of particular genes.
DNA Microarray
Affymetrix
Microarray is a tool for
analyzing gene expression
that consists of a glass slide.
Each blue spot indicates the location of a PCR
product. On a real microarray, each spot is
about 100um in diameter.
DNA Microarray
Millions of DNA strands
build up on each location.
Tagged probes become hybridized
to the DNA chip’s microarray.
DNA Microarrays
• An array works by exploiting the ability of a given mRNA
molecule to hybridize to the DNA template.
• Using an array containing many DNA samples in an
experiment, the expression levels of hundreds or thousands
genes within a cell by measuring the amount of mRNA bound
to each site on the array.
• With the aid of a computer, the amount of mRNA bound to
the spots on the microarray is precisely measured,
generating a profile of gene expression in the cell.
An experiment on a microarray
In this schematic:
GREEN represents Control DNA
RED represents Sample DNA
YELLOW represents a combination of Control and Sample DNA
BLACK represents areas where neither the Control nor Sample DNA
Each color in an array represents either healthy (control) or diseased (sample) tissue.
The location and intensity of a color tell us whether the gene, or mutation, is present in
the control and/or sample DNA.
10
Forensics:
DNA technology in action
• Each of us is genetically unique, with the exception of identical
(monozygotic) siblings. While phenotypic differences are apparent
among us, the most fundamental expression of our uniqueness is in
our genetic material, DNA.
• Today, individuals can be identified through a technique known as
DNA profiling.
• The amount of DNA needed for DNA profiling is very small because
DNA can be amplified through the polymerase chain reaction (PCR).
• One person’s DNA profile is constant, regardless of the type of cell
used to prepare the profile. A DNA profile prepared from a person’s
white blood cells is identical to that prepared from the same person’s
skin cells or other somatic cells.
• Because DNA molecules are only slowly degraded, DNA profiling
can be carried out on biological samples from crime scenes from
years ago, and this profiling has led to the solution of many ‘cold
cases’ worldwide.
Forensic Identification
• Identification using DNA is a powerful tool that can be
applied in many situations including:
– forensic applications
• Can the DNA found at a crime scene be matched to a person on the
national DNA database?
• Is this blood spot from the victim or from the possible assailant?
• In a rape case, is this semen from a previously convicted rapist?
– mass disasters, such as passenger aircraft crashes, the 9/11
terrorist attacks, the Bali bombings
• Can the various remains that have been recovered be matched to a
particular person known to have been on-site?
– identification of human remains
• Are these remains those of a particular missing person?
• Who was the unknown child, tagged as body number 4, recovered
after the sinking of the Titanic in 1912?
What DNA is used for identification
• Depending on the purpose and circumstances of the
identification, the DNA used comes from either the
chromosomes (nuclear DNA) or from mitochondria
(mtDNA).
• In both cases the identification depends on the existence
of segments of DNA that vary greatly between
individuals. Such regions of DNA are termed
hypervariable.
• Hypervariable regions of DNA that are currently used for
identification are:
– short tandem repeats (STRs) in the nuclear DNA, also known
as microsatellites.
– hypervariable regions (HVRs) in the non-coding region of
mtDNA.
STRs and HVRs
Short Tandem Repeats (STRs) in the nuclear DNA, also known as microsatellites.
• A large number of STRs are present on different human chromosomes.
• DNA from STRs can identify one person uniquely (apart from identical siblings).
• DNA samples from relatives are not required.
• Used when there is a need to match a DNA sample from a crime scene to just one
particular person.
Hypervariable Regions (HVRs) in the non-coding region of mtDNA.
• mtDNA identification is less precise because persons from the same maternal line
have identical mtDNA profiles.
• mtDNA is used only when chromosomal DNA cannot be recovered or when
chromosomal DNA is degraded because of age.
• Identification using mtDNA is mainly applied either
– to identify victims of mass disasters where the names of the victims are known but
where identification of the remains by conventional means, such as visual inspection or
dental records, cannot be done, or
– to identify decomposed remains when the identity is suspected to be one of a few
particular missing persons. In both cases, there must be living relatives on the maternal
line to provide mtDNA for comparison with the mtDNA from the remains.
DNA Fingerprinting - HVRs
•
•
•
•
The original technique of identification using DNA
was called DNA fingerprinting and was
developed as an identification tool in 1985 by
Professor Sir Alec Jeffreys
This technique used DNA from hypervariable
regions, known as minisatellites, that are located
near the ends of chromosomes. Minisatellites are
chromosomal regions where sequences of 9 to 80
base pairs are repeated tens or hundreds of
times.
DNA fingerprinting involved cutting minisatellites
from the chromosomal DNA with a restriction
enzyme (Hin fI), separating the DNA fragments by
electrophoresis, transferring them to a membrane
using Southern blotting and exposing the
fragments to one probe that hybridised to a base
sequence present in all the minisatellites.
This probe, known as a multi-locus probe, carried
a radioactive label. The final result seen on an
autoradiograph was a pattern of up to 36 bands,
something like a barcode, with each band being
one allele of one of the minisatellites. Because of
the variation between individuals, each DNA
fingerprint is unique.
The figure above shows the simplified
DNA fingerprints of two people based
on just four hypothetical minisatellites.
In actual DNA fingerprinting, the pattern
for each individual has many more
bands.
DNA profiling - STRs
• DNA fingerprinting has been replaced by a technique known as
DNA profiling that uses short tandem repeats (STRs). These are
• STRs are hypervariable regions of chromosomes where sequences
of just two to five base pairs are repeated over and over. These
regions are very common and hundreds are scattered throughout
the human chromosomes.
• STRs are termed ‘short’ because the repeat sequences are only 2 to
5 base pairs long, and ‘tandem’ because the repeats occur one after
the other. However, the number of repeats at an STR locus can
vary between people and each variation is a distinct allele.
• The number of repeats of a 4-base pair sequence at one STR locus
on the number-5 chromosome ranges from 7 to 15.
• In most cases, the alleles at an STR locus on a human chromosome
are named according to the number of repeats and so are identified
as allele 7, allele 8 and so on. The figure below shows an STR with
7 repeats of the sequence CATT.
DNA profiling - STRs
•
•
•
At each STR locus, one individual is either homozygous or heterozygous
and so can have a maximum of just two different alleles. These alleles are
inherited in a Mendelian fashion.
The figure below shows that a person who is heterozygous 5/7 at one
particular STR locus has one allele with 5 repeats and another allele with 7
repeats.
Within the gene pool of a population, however, many different alleles can
exist at each STR locus.
Frequencies of the alleles at the D5 STR locus on the number-5
chromosome for three sample populations
in Australia.
Note that the allele frequencies vary within a population, with allele 11 being far
more common than allele 15.
Note also that the frequencies vary between populations, with allele 7 being about 20
times more common in Asian populations than in the other two populations.
Why use STRs rather than
minisatellites?
• Compared with DNA fingerprinting, DNA profiling:
– is far more sensitive and requires smaller quantities of DNA (even a
pinhead sized spot of blood can provide sufficient DNA) and the STRs
can be amplified by the polymerase chain reaction (PCR)
– is based on alleles whose sizes allow fragments differing by just one
base pair to be distinguished
– is carried out in a much shorter time — hours rather than days
– uses several single-locus probes rather than one multi-locus probe
– uses coloured fluorescent labels to visualise the STRs rather than
radioactive labels so that each different STR allele can be identified by
colour as well as by size
– produces less complex patterns that are more easily interpreted
• In addition, unlike minisatellites, population data on allele
frequencies of STR alleles can be obtained.
DNA Profiling in Australia
• All Australian states use a common method of DNA
profiling for forensic purposes that involves nine STRs
from different human chromosomes.
• These STR markers were chosen for this purpose
because they are reproducible and robust, easy to score,
are highly informative and have low mutation rates.
• In addition, a tenth marker (that is not an STR) is used to
identify the gender of the individual. This gender marker
is the Amel locus that is present on both the X
chromosome and the Y chromosome.
• The Amel gene on the X chromosome is just 107 base
pairs long while that on the Y chromosome contains 113
base pairs. As a result, the gender of a person can be
identified from this marker.
Loci currently used for DNA
profiling in Australia
For simplicity, STR loci that start with the letter D are identified by their chromosomal location
only, for example, D13 or D7. In reality, the naming of STRs is more complex because there are
multiple STR regions on the one chromosome and these two STRs (D13 and D7) are formally
identified as D13S317 and D7S820.
STR Profiling
•
•
•
To produce a DNA profile, multiple copies of the alleles at these nine STRs
are simultaneously produced using the polymerase chain reaction and the
various alleles are then separated and made visible with fluorescent dyes.
The resulting DNA profile is a series of coloured peaks at different locations,
with each peak being one allele of one specific STR. The location of each
peak indicates the size of the allele and hence the number of repeats.
Where sizes overlap, alleles of different STRs are distinguished by
fluorescent labels of different colours.
STR Profiling
•
•
A person shows either
one or two peaks at
each STR loci, where a
peak corresponds to an
allele, depending on
whether the individual is
homozygous or
heterozygous at that
locus.
For the Amel gender
marker, if just a single
peak with a size of 107
base pairs appears on
the profile, the person is
female; if two peaks are
detected, one at 107
and the second at 113
base pairs, then the
person is male.
This person is female.
They are heterozygous for loci D3, vWA, FGA, D18 and D7.
They are homozygous for loci D8, D21, D5 and D13.
Is STR Profiling reliable?
•
•
STR loci generate many different genotypes (profiles)
For one gene locus with n different alleles, the number of different
genotypes possible is:
[n x (n + 1)]/2.
•
•
•
•
An STR locus with 14 alleles can produce 105 different genotypes in a
population and a different STR locus with nine alleles can have 45 different
genotypes. Together, these two STR loci produce 105 × 45 = 4725 different
genotypes.
As the number of STR loci increases, the number of different genotypic
combinations in a population increases enormously.
As a result, a DNA profile based on nine STRs will be a unique combination
that allows a person to be identified with a very high level of probability.
The chance that the DNA profile of one person will be identical with that of
another person (except for an identical sibling) is one in many billions.
Genetic Engineering
•
•
•
•
•
•
Genetic engineering refers to scientific methods for the artificial
manipulation of genes
Since these methods involve the ‘recombining’ of DNA from different
individuals and even different species, it is often referred to as recombinant
DNA technology
Genetic engineering was made possible by the discovery of a number of
techniques and tools during the 1970s and 1980s
Restriction enzymes can be used to cut DNA (from different sources) into
pieces that are easy to recombine in a test tube
Methods were developed to insert the recombinant DNA into cells, by using
so-called vectors – self-replicating DNA molecules that are used as carriers
to transmit genes from one organism to another
Organisms such as bacteria, viruses and yeasts have been used to
propagate recombinant genes and/or transfer genes to target cells (cells
that receive the new DNA)
Gene Cloning
• Gene cloning is a process of making large quantities of a desired
piece of DNA once it has been isolated
• Cloning allows an unlimited number of copies of a gene to be
produced for analysis or for production of a protein product
• Methods have been developed to insert a DNA fragment of interest
(e.g. a segment of human DNA) into the DNA of a vector, resulting
in a recombinant DNA molecule or molecular clone
• A vector is a self-replicating DNA molecule (e.g. plasmid or viral
DNA) used to transmit a gene from one organism into another
• All vectors must have the following properties:
– Be able to replicate inside their host organism
– Have one or more sites at which a restriction enzyme can cut
– Have some kind of genetic marker that allows them to be easily
identified
• Organisms such as bacteria, viruses and yeasts have DNA which
behaves in this way
• Large quantities of the desired gene can be obtained if the
recombinant DNA is allowed to replicate in an appropriate host
Gene cloning using plasmids
•
Plasmid vectors, found in bacteria, are prepared for
cloning in the following manner:
1.
2.
3.
4.
5.
6.
7.
A gene of interest (DNA fragment) is isolated from human tissue cells
An appropriate plasmid vector isolated from a bacterial cell
Human DNA and plasmid are treated with the same restriction
enzyme to produce identical sticky ends
DNAs are mixed together and the enzyme DNA ligase used to bond
the sticky ends
Recombinant plasmid is introduced into a bacterial cell by simply
adding the DNA to a bacterial culture where some bacteria take up
the plasmid from the solution
The actual gene cloning process (making multiple copies of the
human gene) occurs when the bacterium with the recombinant
plasmid is allowed to reproduce
Colonies of bacteria that carry the recombinant plasmid can be
identified by a genetic marker such as ampicillin resistance
Gene cloning using plasmids
Using bacteria to make proteins for
human use
Gene cloning using viruses
•
Some bacteriophages are convenient for cloning large fragments
of DNA (15 to 20kbp)
•
Main steps in preparing a clone using viral vectors:
1.
2.
3.
4.
5.
6.
7.
8.
A gene is isolated from human tissue cells
An appropriate bacteriophage vector is selected that is capable of
infecting the target cell
Human and the viral DNA are cut with same restriction enzyme
DNAs are mixed together and the enzyme DNA ligase used to
bond the sticky ends
The recombinant DNA is packaged into phage particles by being
mixed with page proteins
The assembled phages are then used to infect a bacterial host
cell
The viral genes and enzymes cause the replication of the
recombinant DNA within the bacterial host cell
The bacterial host cell succumbs to the viral infection. The cell
ruptures (lysis) and thousands of phages, each with recombinant
DNA, are released to infect neighbouring bacteria.
Gene cloning using viruses
Transgenesis
• Trangenesis, using genetic engineering techniques, is concerned
with the movement of genes from one species to another
• An organism that develops from a cell into which foreign DNA has
been introduced is called a transgenic organism
• Because of their immense economic importance, plants have been
the subject of traditional breeding programmes aimed at developing
improved varieties
• Recombinant DNA technology now allows direct modification of a
plant’s genome allowing traits to be introduced that are not even
present in the species naturally
• DNA can now be introduced from other plant species, animals or
even bacteria
• Micropropagation techniques allow introduced genes to become par
of the germ line for plants (the trait is inherited)
• Animal cells may become transformed (receive foreign DNA) to
provide new enhanced characteristics in livestock as well as
providing a means of curing genetic defects in humans through gene
therapy
Transformation using a plasmid
• Ti plasmid isolated from bacteria Agrobacterium
tumefaciens. Agrobacterium tumefaciens causes
tumours (galls) in plants.
• The Ti plasmid can be succesfully transferred to plant
cells where a segment of its DNA can be integrated into
the plant’s chromosome.
• Restriction enzyme and DNA ligase splice the gene of
interest into the plasmid as discussed previously for
cloning into plasmids
• Introduce plasmid into plant cells
• Part of the plasmid containing the gene of interest
integrates into the plant’s chromosomal DNA
• Transformed plant cells are grown by tissue culture
Transformation using a plasmid
Transformation by protoplast
fusion
• This process requires the cell walls of
plant to be removed by digesting enzymes
• The resulting protoplasts (cells that have
lost their cell walls) are then treated with
polyethylene glycol (PEG) which causes
them to fuse
• In the new hybrid cell, the DNA derived
from the 2 “parent” cells may undergo
natural recombination (they may merge)
Transformation by protoplast fusion
Transformation using a gene gun
• This method of introducing foreign DNA
into plant cells, literally shoots it directly
through cell walls using a “gene gun”
• Microscopic particles of gold or tungsten
are coated with DNA and propelled by a
burst of helium through the cell wall and
membrane
• Some of the cells express the introduced
DNA as if it were their own
Transformation using a gene gun
Transformation using liposomes
• Liposomes are small spherical vesicles made of
a single membrane. They can be made
commercially to precise specifications
• When coated with appropriate surface
molecules, they are attracted to specific cell
types in the body
• DNA carried by the liposome can enter the cell
by endocytosis or fusion
• They can be used to deliver genes to these cells
to correct defective or missing genes
Transformation using liposomes
Transformation using viral
vectors
• Some viruses are well suited for gene therapy –
they can accommodate up to 7.5kbp of inserted
DNA in their protein capsule
• When viruses infect and reproduce inside the
target cells, they are also spreading the
recombinant DNA gene
• A problem with this method involves the host’s
immune system reacting to and killing the virus
• Common viruses used for viral transformation of
target cells are retroviruses, lentiviruses and
adenoviruses
Transformation using viral vectors
Transformation using
microinjection
• DNA can be introduced directly into an animal
cell (usually an egg cell) by microinjection
• This technique requires the use a glass
micropipette with a diameter that is much
smaller than the cell itself – the sharp tip can
then be used to puncture the cell membrane
• The DNA is then injected through it and into the
nucleus
Transformation using
microinjection
Making an artificial gene
•
Biologists get genes for cloning from two main sources
– DNA isolated directly from an organism
– complementary DNA (cDNA) made in the laboratory from mRNA templates
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One problem with cloning DNA directly from an organism’s cell is that it
often contains long non-coding regions called introns
These introns can be enormous in length and cause problems when the
gene as a whole is inserted into plasmids or viral DNA vectors for cloning:
– Plasmids tend to lose large inserts of foreign DNA
– Viruses cannot fit the extra long DNA into their protein coats
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To avoid this problem, it is possible to make an artificial gene that lacks
introns
This is possible by using the enzyme reverse transcriptase which is able to
reverse the process of transcription
The important feature of this process is that mRNA has already had the
introns removed, so by using them as the template to recreate the gene, the
cDNA will also lack the intron region
Gene Therapy
• By using the techniques of recombinant DNA technology, medical
researchers attempt to insert a functional gene into a patient’s
somatic cells
• This should make the patient capable of producing the protein
encoded by that allele
• Genetic material delivered to a patient’s cells could be used to treat
a number of conditions:
– Restore the function of a gene that has been lost as a result of a
mutation (i.e. possesses a harmful allele)
– Kill abnormal cells such as those in cancerous tumours
– Introduce genes that inhibit the reproduction of infectious agents such
as viruses, bacteria and endoparasites
– Render cells resistant to toxic drugs used in the medical treatment of
diseases
• By replacing missing genes or modifying faulty genes, it may be
possible to treat genetic diseases
• There have been suggestions that the techniques of gene therapy
may also be put to use to create “designer babies” that have traits
that are selected by the parents
Gene Therapy
• Genetic disorders that are currently undergoing clinical trials include:
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SCIDS
Cancers (including melanoma, breast and colon)
Cystic fibrosis
Haemophilia
Rheumatoid arthritis
Peripheral vascular disease
Inherited high blood cholesterol
• First attempt at gene therapy was when Ashanti DeSilva was treated
for adenosine deaminase (ADA) deficiency on 14 September 1990
• She received new infusions of ADA restored cells every 1-2 months
for the first year, then every 3-6 months thereafter.
• Ashanti is not completely cured - she still takes a low dose of PEGADA. Normally the dose size would increase with the patient's age,
but her doses have remained fixed at her four-year-old level. It's
possible that she could be taken off the PEG-ADA therapy entirely,
but her doctors don't think it's yet worth the risk.
• The fact that she's alive today-let alone healthy and active-is due to
her gene therapy, and also helps prove a crucial point: genes can be
inserted into humans to cure genetic diseases.
Gene Therapy
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In contrast, eighteen-year-old Jesse Gelsinger died on September 17th,
1999 while enrolled in gene therapy trial.
Jesse Gelsinger was not sick before died. He suffered from ornithine
transcarbamylase (OTC) deficiency, a rare metabolic disorder, but it was
controlled with a low-protein diet and drugs, 32 pills a day.
He was not expecting that he would benefit from the study, its purpose was
to test the safety of a treatment for babies with a fatal form of his disorder.
Still, it offered hope, the promise that someday Jesse might be rid of the
cumbersome medications and diet so restrictive that half a hot dog was a
treat. "What's the worst that can happen to me?" he told a friend shortly
before he left for the Penn hospital, in Philadelphia. "I die, and it's for the
babies."
The researchers had tested their vector, at the same dose Jesse got, in
mice, monkeys, baboons and one human patient, and had seen expected,
flulike side effects, along with some mild liver inflammation, which
disappeared on its own.
When Jesse got the vector, he suffered a chain reaction that the testing had
not predicted – jaundice, a blood-clotting disorder, kidney failure, lung
failure and brain death. It is thought that the adenovirus triggered an
overwhelming inflammatory reaction -- in essence, an immune-system
revolt.