Biology Paper 2 - 6.3 Manipulating Genomes

Restriction enzymes are used to cut out DNA fragments. They cut DNA at specific palindromic base sequences (by hydrolysis). Leave sticky or blunt ends.

The sequences consist of short antiparallel base pairs (about 4-6 base pairs). The base pairs read the same in opposite directions.

When restriction enzymes cut in a staggered way leaving small tails of unpaired bases at each end of the fragment. These unpaired bases can be used to bind (anneal) to another DNA fragment that has sticky ends with complementary sequences.

A gene(s) from one organism is isolated and placed into another organism using a suitable vector.

A carrier – transfers DNA into a cell.

They can be a plasmid (small, circular molecules of bacterial DNA) or bacteriophages (viruses that infect bacteria).

The DNA fragment containing the desired gene is isolated using restriction enzymes.
The vector (usually a plasmid) is cut open using the same restriction enzyme.
In this way the sticky ends of the DNA fragment & the vector are complementary.
DNA ligase anneals the vector and DNA fragment (ligation) – forming recombinant DNA.
A plasmid vector then transfers the gene into the bacteria using electroporation.
(a bacteriophage vector will infect the bacterium by injecting its DNA into it & integrate the phage DNA into the bacterial DNA)

A high voltage pulse of electricity is applied to the host (bacterial) cell to disrupt the membrane – making it more porous to encourage uptake of plasmid vector.

Recombinant DNA.

A transformed cell/organism.

Polymerase chain reaction.

A DNA fragment (often containing the desired gene) can be amplified to produce millions of copies in just a few hours.

a) A reaction mixture is set up containing the DNA sample, free DNA nucleotides, primers and DNA polymerase.
b) Mixture heated to 95°C – to separate the DNA strands by breaking the hydrogen bonds, resulting in two template strands.
c) Mixture cooled to 55°C – primers to anneal (join) to each template strand.
d) Mixture heated to 72°C – DNA polymerase joins the lined up complementary nucleotides alongside each template strand to form two new complementary DNA
strands.
e) Cycle starts again, this time all four strands (two original & two new) are used as templates.
f) The cycle is repeated many times, each cycle doubles the amount of DNA.

It does not denature at the high temperatures used in PCR.

PCR amplifies small samples of DNA and so is useful to increase the amount of DNA available for analysis.

An electrical current is used to separate out DNA fragments, RNA fragments or proteins depending on their size.

Cover the agarose gel with a buffer solution.
Fluorescently labelled DNA samples have loading dye added to help them sink to the bottom of the wells in the gel.
Add DNA samples to wells at the negative electrode end.
Pass an electrical current through the gel for about 30 minutes.
The smaller the fragment the faster it travels through the gel.

To provide ions that carry a current and to maintain the pH at a relatively constant value.

DNA is negatively charged and so moves toward the positive electrode (anode) at the other end of the gel.

For visualisation of DNA samples under UV light after electrophoresis to ascertain how far the bands of DNA have travelled.

To compare a set of known banding patterns to identify the length of sample DNA fragments.

RNA is negatively charged and so treated the same as DNA.
Proteins can be positive or negatively charged. They must be denatured before electrophoresis so they are all the same charge.

The fact that the DNA of every individual, except identical twins, is unique.
Within the introns are repetitive, non-coding base sequences called short tandem repeats (STRs).
The number of times these sequences are repeated differs from person to person, so the length of these sequences differs from person to person.

DNA profiling compares the number of times a STR sequence is repeated AND the different places it is repeated in an individual’s genome.
The probability of two individuals having the same length of repetitive sequence is very low.
In modern DNA profiling, 13 STRs are analysed simultaneously.

Sample of DNA obtained (saliva/blood), cut using restriction enzymes (to cut out the exons) and amplified by PCR.
Fluorescent tag added to DNA fragments.
Fragments undergo electrophoresis. Viewed under UV light.
Compare to known banding patterns to identify the length (DNA ladder).

Forensic science.
Preimplantation genetic haplotyping (PGH) – screen embryos for cystic fibrosis & Huntingtons disease before implantation in the uterus.
Paternity disputes.

They do not contain a nucleus and hence have no DNA to profile.

Used to determine the order of bases in a section of DNA (gene) (up to 750 base pairs long). Carried out by chain termination method (Sanger method).

The following mixture is added to 4 separate tubes:
A single-stranded DNA template (the DNA to be sequenced).
DNA polymerase (enzyme that joins DNA together).
Lots of DNA primer (short pieces of DNA) – to start process of DNA synthesis. Also carry a fluorescent labelled tag for identification of DNA fragment produced.
Fluorescently-labelled modified nucleotide –they cannot join to normal nucleotides so acts as chain terminators, ending DNA synthesis.
Free DNA nucleotides + one of the 4 modified nucleotides.
The binding of the (modified) terminator nucleotides (dideoxy nucleotides) is random resulting in strands of varying in lengths.
These strands can be sorted by size using gel electrophoresis.

From the smallest fragment (bottom or positive electrode) to the largest fragment.

It will be complementary to the sequence of the new strand deduced by the chain termination method.

It has become automated and is faster. Instead of running a gel and determining the sequence from that, you get a computer readout.

Faster, cheaper and more accurate (as there is less human error).
Can be done on a large scale (Can screen 1000’s genes at once).
Whole genome sequencing now possible.

The number of additions that occurred when a particular nucleotide was added (bottom).

Sequenced genes and whole genome sequences can be compared between organisms of different species and between organisms of the same species.

Genetic engineering involves the direct transfer of DNA from one organism to another.
Synthetic biology is created from scratch.

Use computers to study biology, e.g create computer simulations & mathematical models.

Developing & using computer software that can analyse, organise & store biological data.

Where you alter defective genes to treat a genetic disorder or cancer.

Gene silencing – where a gene is switched off by putting a piece of DNA inside the gene to stop it from working (used with defective dominant alleles).
Gene supplementation – where the healthy gene is added alongside the faulty one (used with defective recessive genes).

Germ-line therapy - replacing/supplementing the defective gene in the fertilised egg or sex cells (will affect all cells & any offspring produced from these cells).
Somatic-cell therapy - replacing/supplementing the defective the defective gene in body cells that are most affected (needs regular treatment as cells die).

The body could identify vectors as foreign bodies and start an immune response against them.
An allele could be inserted into the wrong place in the DNA, possibly causing more problems, e.g. cancer.

It could prolong the lives of people with genetic disorders and cancer & increase their quality of life.
Carriers of genetic disorders might be able to conceive a baby without that disorder or risk of cancer (only in germ line therapy).

• They will reduce the amount of chemical pesticides used on crops which can harm the environment.

• Encourages monoculture, which decreases biodiversity & whole crop is vulnerable to disease as all genetically identical.

Pharmaceuticals (medicinal drugs) that are produced using GM organisms.

• Drugs can be made in large quantities compared to other methods of production – making them more available to more people.

• There are concerns that manipulating an animal’s genes could cause harmful side effects for the animal – an animal welfare issue.

• Previously untreatable diseases can now be treated, reducing suffering.

• The scientists working with the live pathogen could become infected.

• The GM version of the disease could revert back to its original form.
  Both of these negatives above could cause an outbreak of disease.

• The owner of the patent will get money generated from selling the product – this encourages scientists to genetically engineer products faster.

• Poor farmers may not be able to afford patented GM seeds. Even if they can afford seeds for one year they are not legally allowed to plant & grow any seeds from that crop without paying again – this is unfair to farmers in poorer countries.

Bt gene codes for a protein that is toxic to some of the insects that feed on soybean plants.

Bt gene is isolated using restriction enzymes. Bt gene inserted into plasmid from the bacterium Agrobacterium tumefaciens. Plasmid returned to A. tumefaciens. Soybean plant cells are deliberately infected with the transformed bacteria. The desired gene gets inserted into the soybean plant cells’ DNA – now is GM.

They reduce the amount of chemical pesticides used by farmers which can harm the environment.

May encourage monocultures – where only one type of crop is planted. Monocultures decrease biodiversity & as all plants are genetically identical all susceptible to same diseases.
Bt is toxic to monarch butterflies – but as they only feed on milkweed, so far numbers are unaffected.

GM soya that is resistant to ‘Round up ready’ herbicide so can be grown using this herbicide.
In this way any weeds competing with the soya plants would be killed, but soya plants unharmed.

Environmentalists are concerned about genetic pollution – the potential for the herbicide-resistant gene to pass into weeds, creating superweeds.
Humanitarians are concerned that the main advantage is to the production companies making the herbicide. Poor farmers had to buy both the herbicide-
resistant soya & the herbicide ‘ Round up’ from Monsanto.

Nutritionally enhanced GM rice that contains a gene from daffodils, so that the rice contains beta-carotene.
In India, lack of beta-carotene (the precursor to Vitamin A) causes blindness & sometimes death. As rice is staple diet in this region, GM rice seemed a good
solution.

Humanitarians originally concerned that poor farmers had to buy the seed every year – companies have reacted by giving free licences to farmers to keep & replant rice seeds each year.
Environmentalists concerned about genetic pollution to wild plants.