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Genomes and

gene technologies

By Daniella Di-Fonzo

Specification

Candidates should be able to:

(a) outline the steps involved in sequencing the genome of an organism;

(b) outline how gene sequencing allows for genome-wide comparisons between individuals and
between species (HSW7b);

(c) define the term recombinant DNA;

(d) explain that genetic engineering involves the extraction of genes from one organism, or the
manufacture of genes, in order to place them in another organism (often of a different species) such
that the receiving organism expresses the gene product (HSW6a);

(e) describe how sections of DNA containing a desired gene can be extracted from a donor organism
using restriction enzymes;

(f) outline how DNA fragments can be separated by size using electrophoresis (HSW3);

(g) describe how DNA probes can be used to identify fragments containing specific sequences;

(h) outline how the polymerase chain reaction (PCR) can be used to make multiple copies of DNA
fragments;

(i) explain how isolated DNA fragments can be placed in plasmids, with reference to the role of
ligase;

(j)

state other vectors into which fragments of DNA may be incorporated;


(k) explain how plasmids may be taken up by bacterial cells in order to produce a
transgenic microorganism that can express a desired gene product;
(l) describe the advantage to microorganisms of the capacity to take up plasmid DNA
from the environment;
(m) outline how genetic markers in plasmids can be used to identify the bacteria that
have taken up a recombinant plasmid;
(n) outline the process involved in the genetic engineering of bacteria to produce human
insulin;
(o) outline the process involved in the genetic engineering of Golden RiceTM (HSW6a);
(p) outline how animals can be genetically engineered for xenotransplantation (HSW6a,
6b);
(q) explain the term gene therapy;
(r) explain the differences between somatic cell gene therapy and germ line cell gene
therapy;
(s) discuss the ethical concerns raised by the genetic manipulation of animals (including
humans), plants and microorganisms (HSW4, 6a, 6b, 7c).

Sequencing a genome
Genome

can mean all genes of an individual organism


or all genes in a population of organisms.
Gene technologies used to study genes and their
functions include:

Polymerase chain reaction (PCR)

Cutting out DNA fragments using restriction


enzymes

Gel electrophoresis

DNA probes

Sequencing a Genome
If

a whole genome needs sequencing: use PCR to make multiple copies of all the DNA.
Squeeze through a tiny hole under high pressure. Cuts DNA into 2000- 10000 base
pairs. These can be shortened to make it easier to sequence them.
Make multiple labelled copies of these lengths of DNA. They are mixed with a primer
(up to 20 base pairs of DNA, complimentary to the start of the DNA fragment you
want), free nucleotides, DNA polymerase (to create new DNA strands) and
nucleotides labelled with fluorescent dye. Each base has its own colour and each
labelled nucleotide with stop the chain from growing.
The DNA polymerase attaches free nucleotides to the lengths of DNA. Most of the
copies wont be complete due to the labelled nucleotides. This means lots of different
length chains.
This is then separated using electrophoresis so the DNA is drawn towards the positive
end of a capillary tube.
A computer records the colours as they pass, the shorter the chain, the faster it
travels, so if there are enough fragments, the computer will be able to work out the
sequence of bases in that particular length of DNA.
This is then done for every piece of DNA and then a computer program works out the
overall sequence of nucleotides in the whole genome.

PCR
PCR can be used to make millions of copies of a DNA
fragment in just a few hours.
1.

2.

3.

DNA is denatured by heating to 95C, to break


hydrogen bonds of the double helix of DNA, so the
base pairs are exposed.
The mixture is then cooled to between 50C - 65C
so the primer (up to 20 pieces of DNA
complimentary to the start of the DNA fragment
you want) can bind (anneal) to the strands.
The mixture is then heated to 72C so DNA
polymerase can DNA strand.

5.

6.

7.

8.

The DNA polymerase lines free


nucleotides alongside each
template strand, base pairing
means new complementary
strands are formed.
Two new copies of each DNA
strand are formed from one cycle
of PCR.
The cycle starts again with the
mixture being heated to 95C and
all four strands are used as
templates.
Each PCR cycle doubles the
amount of DNA from the previous
cycle.

Electrophoresis
This separates different DNA fragments according to their lengths.
A

tank containing pure agar (agarose gel) is set up.


A direct current is passed through the gel and the
DNA fragments (which carry a small negative
charge) are attracted to the anode (positive
electrode)
The smaller the fragment, the faster they move.
Labelled nucleotides allow us to use time to sort
longer DNA strands from shorter ones.

Electrophoresis (cont.)
Probes form hydrogen
bonds with stretches of
DNA close to its
complimentary
sequence

We

Used for DNA


profiling.
Phosphurus 32 is
the radioactive
isotope

can also use distance to sort the strands.


When the current is turned off, the DNA fragments have ended up in
different places and arent immediately visible.
Transfer them onto absorbent paper placed on top of the gel and heat so
the two DNA strands in each molecule separate from each other.
Short sequences of single stranded DNA are added, these are labelled
using fluorescents or radioactive isotopes.
These pair up with the DNA fragments on the paper and can be detected
using UV light or X-ray film.
Then we can match the marks to the positions of DNA fragments on the
agarose gel.

Restriction enzymes
You can get a DNA fragment from a length of DNA using
restriction enzymes.
1.
2.

3.

4.
5.

Cut DNA backbones


Restriction nucleoases that cut DNA at specific
points.
Each particular enzyme cuts DNA at a different
point- (the restriction site)
Less than 10 bases long.
Catalyse a hydrolysis reaction that breaks the
phosphate sugar backbone of the double helix.
Leaves exposed bases called sticky ends.

DNA ligase
Catalyses

condensation reaction that joins the


phosphate- sugar backbones in the helix together.
Both pieces of DNA need to have been cut with the
same restriction enzyme, so the sticky ends are
complimentary and can hydrogen bond together.
DNA ligase then seals the backbone.
The resulting DNA is recombinant DNA.

Genetic engineering
Using

technology to change
genetic material of an
organism.
DNA containing lengths from
two different species is called
recombinant DNA
Organisms with altered DNA
are transformed organism
Genes can be manufactured
instead of extracted
An organism with DNA from
another organism is a
transgenic organism

Ethical issues- some people believe.


1. using antibiotic resistant genes as
markers may increase number of
antibiotic resistant pathogens in our
environment
2. May encourage farmers to
monoculture and decrease biodiversity
and leave the whole crop susceptible
to disease because they are
genetically identical
3. Engineering animals for
xenotransplantation may cause
suffering
4. May produce herbicide resistant weeds
5. Companies may exploit farmers in poor
countries by selling them crops they
cant afford
6. Some people worry humans will be
genetically engineered, e.g., to be
smarter

Human insulin
People

with type 1 diabetes need to inject insulin to regulate their blood glucose
concentration
Used to be extracted from pigs, now its mad by genetically engineered bacteria
1. Gene for human insulin is identified and isolated using restriction enzymes
2. A plasmid is cut open using the same restriction enzyme used to isolate the gene
3. The insulin gene is inserted into the plasmid to make recombinant DNA
4. The plasmid is taken up by bacteria and any transformed bacteria are identified
using marker genes
5. The bacteria are grown in a fermenter and human insulin is produced as bacteria
grow and divide
6. The human insulin is extracted and purified
.Advantages
1. More effective than animal insulin and less chance of rejection because its
identical to human insulin
2. Cheaper and faster to produce, more reliable and larger supply of insulin
3. Overcomes ethical or religious issues arising from using animal insulin

Golden rice
Genetically

engineered to produce beta carotene which is used to


produce vitamin A. Being used to reduce vitamin A deficiency in
places like south Asia and parts of Africa
1. Psy gene is extracted from daffodil and crtl gene extracted from soil
bacterium using restriction enzymes
2. A plasmid is removed from Agrobacterium tumefaciens and cut
open using the same restriction enzymes
3. The genes and a marker gene are inserted into the plasmid
4. The recombinant plasmid is put back into the bacterium
5. Rice plants are incubated with the transformed bacteria which infect
the plant cells
6. Rice plants are grown on a selective medium so only the
transformed plants will be able to grow because they contain the
marker needed to grow on this medium

xenotransplantation

Xenotransplantation is the transplantation of cells, tissues or organs from one


species to another

Animals may be used as organ donors for humans when there is a shortage of
human donors
However, there is a risk of rejection
Scientists are trying to eliminate this risk by:

Inserting genes for human cell surface proteins into a newly fertilised animal embryo.
The genes integrate into the animal DNA lowering the risk of rejection
2)
Genes for animal cell surface proteins are inactivated. Removed from the nucleus of
animal cell and then transferred into an unfertilised animal egg. The egg cell is then
stimulated to divide and the animal created doesnt produce animal cell surface
proteins
e.g., a pig that doesnt have the enzymes needed for it to make a sugar that humans dont
have but pigs usually do.
1)

Gene therapy
Somatic alters
the alleles in
body cells.
Offspring can
still inherit the
disease e.g.,
cystic fibrosis

Two types of
gene therapy

Germ line- alters


the alleles in sex
cells. Offspring
cant inherit the
disease.
Currently illegal
in humans

Gene therapy alters alleles inside


cells to cure genetic disorders. If
its caused by two recessive alleles,
then a dominant allele can be
added to make up for them. If its
caused by a dominant allele, this
can be altered by putting a bit of
DNA in the middle of it.

The allele is inserted into cells


using a vector
Viruses, plasmids or liposomes
are different vectors

Advantages

disadvantages

Prolong lives of people with genetic disorders

Effects may only be short lived (somatic)

Give better quality of life to sufferers

Patient may need multiple treatments


(somatic)

Allow them to have offspring with out


disorder (germ line)

Might be difficult to get allele into specific


body cells

Decrease number of sufferers (germ line)

May be rejection by immune system


Allele inserted in wrong place could cause
problems e.g., cancer
Inserted allele could be over expressed,
producing too much missing protein

There may also be


ethical concerns and
financial concerns.

Disorders caused by multiple genes difficult


to treat this way.

Dna probes
Can

identify DNA fragments that contain specific sequences of


bases, e.g., see if DNA contains mutated gene
Theyre short strands of DNA that have a base sequence
complimentary to the one theyre looking for
Probes have fluorescent or radioactive labels so it can be detected
Can be used to see if any family members have a gene that causes
a genetic disorder
1.

2.

3.
4.

A sample of DNA is digested into fragments using restriction enzymes


and separated using electrophoresis
Transferred to a nylon mb and incubated with probe thats
complimentary to mutated gene
If the mutated gene is present, the probe will bind (hybridise) to it
Mb is then exposed to UV light and if the gene is present, it will fluoresce

Chain termination- determines order of


bases in a section of DNA
1.

2.

3.

4.

Single stranded DNA, primer, DNA polymerase, free nucleotides


and fluorescently labelled nucleotides are mixed together and put
in four tubes. A different modified nucleotide is added to each of
the tubes.
They undergo PCR, which produces many strands of DNA. Theyre
different lengths because they terminate at different points
depending on where the modified nucleotide was added
The DNA fragments are separated by electrophoresis and
visualised under fluorescent light
The complimentary base sequence can be read from the gel. The
smallest nucleotide is at the bottom and by reading the bands from
top to bottom, you can build the DNA sequence one base at a time

Bacterial artificial chromosomes (bacs)


To
1.
2.

3.
4.

5.

6.

7.

sequence an entire genome, you need smaller pieces


Genome is cut into 100 000 bp using restriction enzymes
Fragments are inserted into BACs- man made plasmids. Each
fragment is inserted into a different BAC
The BACs are then inserted into different bacteria
The bacteria divide into colonies of clones with specific DNA
fragments, together they make a complete genomic DNA library
DNA is extracted from each colony and cut up using restriction
enzymes, producing overlapping pieces of DNA
Each piece of DNA is sequenced using chain termination, and the
pieces are put back in order to give the full sequence from that BAC
DNA fragments from all BACs are put back in order to give the entire
genome

Comparing genomes
Comparing genomes of different species

Comparing genomes of the same species

Understand evolutionary relationships.


Closely related species will share more DNA

Trace early human migration. Compare


genomes of people in different parts of the
world

Understand how genes interact during


development and how theyre controlled

Study genetics of human diseases to detect


particular mutations that lead to an increased
risk of a disease

Carry out medical research on genes found in


genomes of other mammals e.g., cancer

Develop medical treatments for particular


genotypes