2. Techniques to Study Genes
Polymerase Chain Reaction (PCR)
Cutting out DNA fragments using Restriction Enzymes
Electrophoresis
Finding specific sequences of DNA using DNA Probes

3. Polymerase Chain Reaction
The PCR amplifies small DNA fragments which can be useful in forensic
investigations. A reaction mixture is set up with the DNA sample, free
nucleotides, primers* and DNA polymerase and heated to 95degC to
break the hydrogen bonds between the two strands of DNA. The
samples are now single stranded so the mixture is cooled to 55degC so
the primers can anneal to the strands. ‘Taq polymerase’* is added to
synthesise two new strands of DNA so it is now double stranded… the
temperature is raised to 72degC so DNA polymerase can bind to this
double strand (complementary base pairing). Two new copies are
formed. This cyclic reaction can be repeated many times.

4. Using Restriction Enzymes
(RE)
Some sections of DNA have palindromic* sequences of
nucleotides; RE recognise specific palindromic sequences and cut
the DNA at these places. Different RE cut at different specific
points because the shape of the recognition sequence is
complementary in shape to the enzyme’s active site. The DNA
sample is incubated with the specific RE, which cuts the DNA
fragment out via a hydrolysis reaction which breaks the sugar-
phosphate backbone. Sometimes, this leaves sticky ends* which
can be used to anneal the DNA fragment to another piece of DNA
that has sticky ends with complementary sequences.

5. Electrophoresis
A flourescent tag is added to all the DNA fragments so they can
be viewed under UV light. The DNA is placed into a well in a slab
of gel and covered in a buffer solution that conducts electricity. An
electric current is passed through the gel – DNA fragments are
negatively charged because of its phosphoryl groups, so they
move towards the positive electrode (anode). Small DNA
fragments move faster and travel further through the gel, so the
DNA fragments separate according to size. The DNA fragments
are viewed as bands under UV light,

6. DNA Probes
A DNA probe is a short single-stranded section of DNA that is
complementary to the section of DNA being investigated. A DNA
probe will hybridise (bind) to the target sequence if it’s present in a
sample of DNA. It has a label attached so it can be detected; the
most common types are a radioactive marker (detected using X-
ray) or a flourescent marker (detected using UV light).
One use of DNA probes are to see if a family member has a
mutation in a gene that causes a genetic disorder.

7. Terms*
Primer  a short piece of DNA that is complementary to the
bases at the start of the fragment you want
‘Taq Polymerase’ an enzyme which is thermophillic so isn’t
denatured by the hot temperatures used during the PCR
Palindromic  antiparallel base pairs (read the same in
opposite directions)
Sticky Ends  small tails of unpaired bases a each end of the
fragment

8. Sequencing the Genome
Genomes are firstly mapped. Samples of the genome are
mechanically sheared into smaller sections and placed in
separate Bacterial Artificial Chromosomes (BACs) and transferred
to E-Coli cells. As the cells grow in culture, many copies are
produced called clone libraries. Cells containing specific BACs are
taken and cultured. The DNA is extracted and RE cut it into small,
separate fragments which are separated during electrophoresis.
This allows genome-wide comparisons…

9. How?
The identification of genes for proteins found in all or many
living organisms gives clues to the relative importance of such
genes to life
Comparing the DNA of different species shows evolutionary
relationships
Modelling the effects of changing DNA can be carried out
Comparing genomes from pathogenic and similar non-
pathogenic organisms identifies the genes that causes the
disease so more effective drugs can be developed

10. Genetically Engineering a
Microorganism
1. The DNA fragment containing the desired gene is obtained
2. The DNA fragment (with the gene in) is inserted into a Vector
3. The Vector transfers the gene into the bacteria
4. Identify the transformed bacteria

11. Steps 1 & 2…
The DNA fragment containing the gene you want is isolated using RE.
the DNA fragment is then inserted into a vector using RE and ligase. A
vector transfers DNA into a cell; they can be plasmids or
bacteriophages. The vector is cut open the same RE that was used to
isolate the DNA fragment containing the desired gene so the sticky ends
of the vector are complementary to the sticky ends of the DNA fragment
containing the gene. The vector DNA and DNA fragment are mixed
together with DNA ligase which joins up the sugar-phosphate backbone
of the two bits (ligation). The new combination of bases in the DNA
(vector DNA + DNA fragment) is called recombinant DNA.

12. Step 3: Transferring the Gene
The vector with the recombinant DNA is used to transfer the gene
into the bacterial cells. If a plasmid vector is used, the bacterial
cells have to be persuaded to take in the plasmid vector and its
DNA (e.g. they’re placed in ice-cold calcium chloride to make their
cell walls more permeable). The plasmids are added and the
mixture is heat shocked which encourages the cells to take in
plasmids. With a bacteriophage vector, the bacteriophage will
infect the bacterium by injecting its DNA into it. The phage DNA
then integrates into the bacterial DNA. Cells that take up the
desired gene are transformed.

13. Step 4: Identifying Transformed
Bacteria
Not all bacteria take up the vector, so those that have can be
identified using genetic markers. The genetic markers are inserted
into vectors at the same time as the desired gene. The bacteria
are grown on agar plates and each cell divides and replicates its
DNA, creating a colony of cells. Transformed cells will produce
colonies where all the cells contain the desired gene and the
marker gene. The marker gene can code for antibiotic resistance,
so the bacteria will be resistant to Ampicillin and Tetracycline. The
agar plates contain the antibiotic so only cells with the marker
gene will survive and grow.

14. Producing Human Insulin
The gene for human insulin is identified and isolated using RE.
Specialised centrifugation methods separate mRNA from pancreatic
tissue and reverse transcriptase uses this mRNA as a template to make
single-stranded cDNA (made double by DNA polymerase). A single
sequence of nucleotides are added to each end of the DNA to make
sticky ends. A plasmid is cut open using the same RE. Plasmids and the
insulin gene are mixed so sticky ends form base pairs. DNA ligase links
the sugar-phosphate backbone of the plasmid and insulin gene making
it recombinant. Plasmids are mixed with bacteria in the presence of
calcium ions so some take up the plasmids and form a clone.
Genetically engineered bacteria transcribe and translate the human
gene to make insulin.

15. Advantages
It is advantageous to genetically engineer human insulin rather
instead of using animal insulin…
• It’s identical to human insulin so it will be more effective and
there will be less risk of a reaction
• It is a cheaper and faster way of providing a more reliable,
larger supply of insulin
• No ethical or religious issues

16. ‘Golden Rice’
The psy gene (from maize) and the crtl gene (from the soil bacterium)
are isolated using RE. A plasmid is removed from the Agrobacterium
tumefaciens bacterium and cut open with the same RE. The psy and crtl
and a marker gene are inserted into the plasmid; this recombinant
plasmid is put back into the bacterium. Rice plant cells are incubated
with the transformed A.tumefaciens bacteria, which infect the rice plant
cells. A.tumefaciens inserts the genes into the plant cells’ DNA creating
transformed rice plant cells. The rice plant cells are then grown on a
selective medium so only transformed rice plants with the marker gene
will be able to grow.

17. Advantages of Golden Rice
The resulting plants produce seeds with beta-carotene in the
endosperm. Our bodies use beta-carotene to produce Vitamin A so
‘Golden Rice’ is being developed in countries such as south Asia and
parts of Africa to help reduce Vitamin A deficiency. Vitamin A is needed
for: bone growth; to form part of rhodopsin, a pigment needed for
eyesight; maintenance and differentiation of epithelial cells which help
reduce infection; synthesis of glycoproteins for cell growth and
development. But, ‘Golden Rice’ has raised ethical issues and has said
to reduce biodiversity by encouraging farmers to carry out monoculture
which would leave the whole crop vulnerable to disease as they are
genetically identical.

18. Gene Therapy
Somatic Cell Gene Therapy (SCGT):
• Augmentation  (adding genes). Some conditions are caused
by the inheritance of faulty alleles leading to the loss of a
functional gene product. Engineering a functioning copy of the
gene into relevant specialised cells means the polypeptide is
synthesised and the cells can function normally
• Killing Specific Cells  genetic techniques make such cells
(e.g. cancers) express genes to produce proteins that make
cells vulnerable to attack by the immune system

19. Gene Therapy
Germline Cell Gene Therapy (GCGT) involved engineering a
gene into the sperm, egg or zygote or into all cells of an early
embryo so as the organism grows, every cell contains a copy of
the engineered gene. Here, the genetic modification is
restricted to somatic (body) cells with no effect on the germline.
An individual can still pass on the allele for the disorder to their
offspring. This gene therapy, although legal, is ethically
unacceptable for fear it could create a new disease or interfere
with evolution.

20. Somatic Germline
The functioning allele of the gene is
introduced into the target cells
The functioning allele is introduced into
germline cells so delivery techniques
are more straight forward
Treatment is short lived and has to be
repeated. The specialised cell will not
divide and pass onto the offspring
All cells derived from the germline will
contain a copy of the functioning allele.
It can be passed onto the offspring
Difficulties in getting the allele into the
genome in a functioning state
It is unethical to engineer human
embryos

21. Issues with Genetic
Engineering
Some people are worried that using antibiotic-resistance genes as
marker genes may increase the number of antibiotic-resistant,
pathogenic microorganisms
Genetically engineering animals for xenotransplantation (transfer of
cells/tissues/organs from one species to another) may cause them
suffering
Some are concerned about ‘superweeds’ – weeds that are resistant to
herbicides as they’ve bred with genetically engineered herbicide-
resistant crops
What is humans are genetically engineered and a genetic underclass is
created? (This is currently illegal)