Genetics & Evolution Series: Set 9 Version: 2.0. Gene technology is a broad field which includes analysis of DNA as well as genetic engineering and other.

Genetics & Evolution Series: Set 9Version: 2.0

Gene technology is a broad field which includes analysis of DNA as well as genetic engineering and other forms of genetic modification.

Genetic engineering refers the artificial manipulation of genes: adding or subtracting genes, or changing the way genes work.

Organisms with artificially altered DNA are referred to as genetically modified organisms (GMOs).

Gene technologies have great potential to benefit humanity through:

increasing crop production

increasing livestock production

preventing and fighting disease

reducing pollution and waste

producing new products

detecting and preventing crime

What is Gene Technology?

Why Gene Technology?

Photos courtesy of GreenPeace

Who owns and regulatesthe GMOs?

Third world economies areat risk of exploitation

Biological risks have notbeen adequately addressed

Animal ethics issues

The costs of errors

Environmentally friendly

Could improve thesustainability of crop andlivestock production

Could potentially benefitthe health of many

More predictable anddirected thanselective breeding

Despite potential benefits, gene technology is highly controversial.

Some people feel very strongly that safety concerns associated with the technology have not been adequately addressed.

The Beginning of GE

The bacterium Escherichia coli (above) and the yeast Saccharomyces cerevisiae (below): favorite organisms

of gene research

Genetic engineering (GE) was made possible by the discovery of new techniques and tools in the 1970s and 1980s.

It builds on traditional methods of genetic manipulation, including selective breeding programs and the deliberate introduction of novel traits by exposing organisms (particularly plants) to mutagens.

Methods were developed to insert ‘foreign’ DNA into cells using vectors. New recombinant DNA technology involved ‘recombining’ DNA from different individuals and even different species.

Organisms such as bacteria, viruses, and yeasts are used to propagate recombinant genes and/or transfer genes to target cells (cells that receive the new DNA).

Producing GMOsGMOs may be created by modifying their DNA in one of three ways:

Adding a Foreign GeneA foreign gene is added which will enable the GMO to carry out a new genetic program. Organisms altered in this way are referred to as transgenic.

Host DNA

Delete or ‘Turn Off’ a GeneAn existing gene may be deleted or deactivated to prevent the expression of a trait (e.g. the deactivation of the ripening gene in tomatoes).

Host DNA

Alter an Existing Gene An existing gene already present in the organism may be altered to make it express at a higher level (e.g. growth hormone) or in a different way (in tissue that would not normally express it). This method is also used for gene therapy.

Existing gene altered

Host DNA

Restriction Enzymes

Recognition SiteRecognition Site

GAATTC

CTTAAG

DNA

CTTAAG

GAATTC

cutThe restriction enzyme

EcoRI cuts here

cut cut

Restriction enzymes are one of the essential tools of genetic engineering. Purified forms of these naturally occurring bacterial enzymes are used as “molecular scalpels”, allowing genetic engineers to cut up DNA in a controlled way.

Restriction enzymes are used to cut DNA molecules at very precise sequences of 4 to 8 base pairs called recognition sites (see below).

By using a ‘tool kit’ of over 400 restriction enzymes recognizing about 100 recognition sites, genetic engineers are able to isolate and sequence DNA, and manipulate individual genes derived from any type of organism.

Specific Recognition SitesRestriction enzymes are named according to the bacterial species they were first isolated from, followed by a number to distinguish different enzymes isolated from the same organism.

e.g. BamHI was isolated from the bacteria Bacillus amyloliquefaciens strain H.

A restriction enzyme cuts the double-stranded DNA molecule at its specific recognition site:

Enzyme Source Recognition Sites

EcoRI Escherichia coli RY13 GAATTC

BamHI Bacillus amyloliquefaciens H GGATCC

HaeIII Haemophilus aegyptius GGCC

HindIII Haemophilus influenzae Rd AAGCTT

Hpal Haemophilus parainfluenzae GTTAAC

HpaII Haemophilus parainfluenzae CCGG

MboI Moraxella bovis GATC

NotI Norcardia otitidis-caviarum GCGGCCGC

TaqI Thermus aquaticus TCGA

It is possible to use restriction enzymes that cut leaving an overhang; a so-called “sticky end”.

DNA cut in such a way produces ends which may only be joined to other sticky ends with a complementary base sequence.

See steps 1-3 opposite:

C T T A A

A A T T C G

G

Sticky Ends

FragmentRestriction

enzyme: EcoRI

Sticky endRestriction enzyme: EcoRI

DNA from

another source

A restriction enzyme cuts the double-stranded

DNA molecule at its specific recognition site

The two different fragments cut

by the same restriction enzyme

have identical sticky ends and

are able to join together

The cuts produce a

DNA fragment with

two “sticky” ends

When two fragments of DNA cut by the same restriction

enzyme come together, they can join by base-pairing

C T T A A

A A T T C

G

G A A T T C

C T T A AG

G

C T T A A

A A T T C G

G

C C C

G G G

G G G

C C C

C C C

G G G

G G G

C C C

C C C

G G G

G G G

C C C

Blunt Ends

It is possible to use restriction enzymes that cut leaving no overhang; a so-called “blunt end”.

DNA cut in such a way is able to be joined to any other blunt end fragment, but tends to be non-specific because there are no sticky ends as recognition sites.

Restriction enzyme

cuts here

Recognition Site Recognition Site

DNA from another source

The cut by this type of restriction

enzyme leaves no overhang

cutcut

C C C

G G G

G G G

C C C

C C C

G G G

G G G

C C C

G G G

G G G

C C C

C C C

DNA

A special group of

enzymes can join

the pieces together

LigationDNA fragments produced using restriction enzymes may be reassembled by a process called ligation.

Pieces of DNA are joined together using an enzyme called DNA ligase.

DNA of different origins produced in this way is called recombinant DNA because it is DNA that has been recombined from different sources.

Steps 1-3 are involved in creating a recombinant DNA plasmid:

Plasmid DNA fragment

Two pieces of DNA are cut using the same restriction enzyme.

Foreign DNA fragment

A A T T C G

C T T A AG

The two different DNA fragments are attracted to each other by weak hydrogen bonds.

This other end of the foreign DNA is attracted

to the remaining sticky end of the plasmid.

When the two matching “sticky ends” come together, they join by base pairing. This process is called annealing.

This can allow DNA fragments from a different source, perhaps a plasmid, to be joined to the DNA fragment.

The joined fragments will usually form either a linear molecule or a circular one, as shown here for a plasmid.

Annealing

Detail of Restriction Site

Restriction sites on the fragments are attracted by base pairing only

Gap in DNA molecule’s ‘backbone’

Foreign

DNA

fragment

A A T TC

A A T T C

G

G

C A

Plasmid

DNA

fragment

G

G

T T A

AATTC

DNA ligase

The fragments are able to

join together under the

influence of DNA ligase.

Recombinant DNA Plasmid

GA A T T

C

G A A T T C

C T T A A G

GAATT

C

Recombinant Plasmid DNA

Detail of Restriction Site

Fragments linked

permanently by

DNA ligase

No break in

DNA molecule

The fragments of DNA are joined together by the enzyme DNA ligase, producing a molecule of recombinant DNA.

These combined techniques of using restriction enzymes and ligation are the basic tools of genetic engineering.

DNA Amplification

A crime scene(body tissue samples)

Fragments of DNA from

a long extinct animal

A single viral particle

(from an infection)

Using the technique called polymerase chain reaction (PCR), researchers are able to create vast quantities of DNA identical to trace samples. This process is also known as DNA amplification.

Many procedures in DNA technology require substantial amounts of DNA to work with, for example;

DNA sequencing

DNA profiling/fingerprinting

Gene cloning

Transformation

Making artificial genes

Samples from some sources,including those shown here,may be difficult to obtain inany quantity.

PCR EquipmentAmplification of DNA can be carried out with simple-to-use PCR machines called thermal cyclers (shown below).

Thermal cyclers are in common use in the biology departments of universities as well as other kinds of research and analytical laboratories.

Steps in the PCR Process

The laboratory process called the polymerase chain reaction or PCR involves the following steps 1-3 each cycle:

Separate StrandsSeparate the target DNA strands by heating at 98°C for 5 minutes

Add Reaction MixAdd primers (short RNA strands that provide a starting sequence

for DNA replication), nucleotides (A, T, G and C) and DNA

polymerase enzyme.

IncubateCool to 60°C and incubate for a few minutes. During this time, primers

attach to single-stranded DNA. DNA polymerase synthesizes complementary strands.

Repeat for about 25 cycles

Repeat cycle of heating and cooling until enough copies of the target DNA

have been produced.

Although only three cycles of replication are shown here, following cycles replicate DNA at an exponential rate and can make literally billions of copies in only a few hours.

The process of PCR is detailed in the following slide sequence of steps 1-5.

Polymerase Chain ReactionPCR

cycles

No. of target

DNA strands

1 2

2 4

3 8

4 16

5 32

6 64

7 128

8 256

9 512

10 1024

11 2048

12 4096

13 8192

14 16 384

15 32 768

16 65 536

17 131 072

18 262 144

19 524 288

20 1 048 576

21 2 097 152

22 4 194 304

23 8 388 608

24 16 777 216

25 33 554 432

Cycle 1

Cycle 2

Cycle 3

Original DNASample

The Process of PCR 1

Primer annealed

A DNA sample called the

target DNA is obtained

DNA is denatured (DNA strands

are separated) by heating the

sample for 5 minutes at 98C

Primers (short strands of mRNA)

are annealed (bonded) to the DNA

The Process of PCR 2Nucleotides

Nucleotides

After one cycle, there are now two copies of the original sample.

The sample is cooled to 60°C.

A thermally stable DNA polymerase enzyme binds to the primers on each side of the exposed DNA strand.This enzyme synthesizes a complementary strand of DNA using free nucleotides.

A technique known as gel electrophoresis can be used to separate large molecules (including nucleic acids or proteins) on the basis of their size, electric charge, and other physical properties.

To prepare DNA for electrophoresis, the DNA is often cut up into smaller pieces. Called a restriction digest, and it produces a range of DNA of different lengths.

To carry out electrophoresis, the DNA samples are placed in wells and covered with a buffer solution that gradually dissolves them into solution.

Gel ElectrophoresisWells into which samples to be analyzed are placed.

Buffer solution

Cathode

Anode Gel

Plastic Frame

Buffer

Sample

DNA fragments, shown symbolically above, move towards the positive terminal (smaller fragments move faster than longer ones).

By applying an electric field to the solution, the molecules move towards one or other electrode depending on the charge on the molecule itself. DNA is negatively charged because the phosphates have a negative charge.

Molecules of different sizes (molecular weights) become separated (spread out) on the gel surface.

These can be visualized by applying dyes or radio-labeled probes.

Analyzing DNA

-ve terminal

+ve terminal

Small fragments

Large fragments

Tray: Contains the set gel.

DNA solutions: Mixtures of different sizes of DNA fragments are loaded into each well.

DNA markers: A mixture of DNA molecules with known molecular weights. They are used to estimate the sizes of the DNA fragments in the sample lanes.DNA fragments:

The gel matrix acts as a seive for the DNA molecules.

Wells: Holes created in the gel with a comb.

DNA ProfilingDNA profiling (DNA fingerprinting) is a technique for genetic analysis, which identifies the variations found in the DNA of every individual.

The profile refers to the distinctive pattern of DNA restriction fragments or PCR products which is used to identify an individual.

There are different methods of DNA profiling, each with benefits and drawbacks.

DNA profiling does not determine a base sequence for a sample but merely sorts variations in base sequences.

Only one in a billion (i.e. a thousand million) persons is likely to have an identical DNA profile, making it a useful tool for forensic investigations and paternity analysis.

DNA fragments (PCR product after endonuclease digestion) visualized under UV light after staining with ethidium bromide and migration in an agarose electrophoresis gel.

Visualizing the Profile

DNA Profiling MethodsDNA profiling begins by extracting DNA from the cells in a sample of blood, saliva, semen, or other fluid or tissue.

Two methods are commonly used. Both are based on the analysis of short repetitive sequences in the DNA.

Profiling using probes (RFLP analysis) was the first profiling technique to be developed. Restriction enzymes are applied to a DNA sample and the DNA fragments are separated on a gel. Radioactive probes are used to label DNA fragments with complementary sequences.

Profiling using PCR is newer technique which uses highly polymorphic regions of DNA that have short repeated sequences of DNA. These sequences are amplified using PCR and then separated on a gel.

This technique is suitable when there is very little DNA available or the sample is old.