Types of cloningDNA Cloning (Recombinant DNA

Technology)

Reproductive Cloning (in animals)

Cloning in agriculture

DNA Cloning reviewBacteria can be transformed with recombinant plasmids; plasmids (carry-ing a specificgene can then be cloned tomake many copies)

Reproductive Cloning

Two types:

1. Somatic nuclear cell transfer

Animals can be cloned by removing the nucleus from a somatic cell and injecting it into an egg cell that has had its nucleus removed (somatic cell nuclear transfer).

After some chemical treatment, the cell starts dividing and develops into an embryo, is implanted into a surrogate mother who carries it to term.

Can be fusion of somatic cell and egg cell or microinjection of only somatic cell nucleus.

Some notes about animal reproductive cloningDolly the Sheep – this was done to determine

if a specialized somatic cell could be re-programmed to produce a new organism

Not a total clone since there is always some mitochondrial DNA that contributes to the genetic make-up of an organism

Reproductive cloning2. Artificial Embryo Twinning

Mimics the natural process of creating identical twins in a Petri dish.

Embryos are separated into individual cells early on; those cells develop into embryos which are carried to terms by surrogate mothers

Therapeutic cloningAlso called ‘embryo cloning’ – the production

of human embryos for use in research (illegal in Canada)

Generating stem cells to grow new tissues and organs

Cloning in agriculture

Tissue culture – small piece of plant grown in test tube, produces

shoots that are identicalOR

Cloning vector – use of a bacterial plasmid, infects plants (Ti plasmid can incorporate into host genome)

Foreign genes can be inserted by using a bacteria, Agrobacterium tumefaciens, that can carry a recombinant plasmid into a plant.

LimitationsA. tumefaciens only infects dicots (e.g.

soybeans, potatoes, tobacco, etc.)

Now, DNA molecules can be injected directly into plant cells (microinjection, electroporation and particle bombardment)

Applications in agriculture

Designing transgenic (also known as genetically

modified) plants. Why?

Resist herbicides (round-up ready corn)Resist insect pests and diseases (Bt corn)Less need for artificial fertilizersResist environmental stressesProduce plants with new characteristics (e.g.

longer shelf life)

Examples of transgenic plants

FlavrSavr tomato

Golden rice

Triple stack corn

Applications in agriculture cont’d

Increasing desirable characteristics in livestock (e.g. cows, sheep, goats, chickens, etc.). Such as?

Increased production of meat, milk, eggs and other animal products

Better disease resistanceChanging the fat:lean ratio of meat

Techniques used in biotechnology research and applications

RFLP analysisRestriction fragment length polymorphism

(RFLP – pronounced rif-lip) is a difference in homologous DNA sequences

RFLP analysis is the original method used for profiling DNA and was used in genetic fingerprinting and genome mapping before cheaper methods such as PCR (polymerase chain reaction) and DNA sequencing came along

How does it work?DNA samples are digested by restriction

enzymesThen the DNA fragments are run on an

electrophoresis gelThen an RFLP probe (radioactive DNA probe –

sequence known) is added and will bind to certain matching bases – this is how they started mapping the human genome

RFLP Analysis

Comparison of different lengths of DNA fragments produced by restriction enzymes to determine genetic differences between individuals or to detect disease

A pioneering method but…..It takes about one month for the process

It requires a large amount of DNA

Still used to detect genetic variation between individuals, for inherited diseases and more generally for disease detection

A quick and easy way to make lots of copies of the DNA we want

Polymerase Chain Reaction (PCR)

Why PCR?Often a gene of interest is not in large

enough quantities to analyze

Much faster than cloning a certain gene or piece of DNA in bacteria

All happens in a test tube:1st step: Denaturation – the break down of double-stranded DNA using

heat2nd step: Annealing – primers attach to either end; lower temperature

3rd step: Extension- DNA polymerase extends the strand using

nucleotides that have been added, medium temperature

And the test tubes are in this……

ApplicationDNA fingerprinting for forensics and

paternity

Genetic testing for hereditary and infectious diseases

Move aside PCRConstant T PCR has been developed

Recombinase polymerase amplification

No need for sophisticated PCR equipment

Agarose Electrophoresis of DNA

used to separate fragments of DNA

DNA is negatively charged, charge is proportional to size

agarose can be used as a molecular strainer (sieve) to separate the pieces of DNA by size

Gel electrophoresis

A current is run through the buffer surrounding the gel and it pushes the DNA away from the negative anode, towards the positive cathode

A current is run through the buffer surrounding the gel and it pushes the DNA away from the negative anode, towards the positive cathode

Gel Electrophoresis

DNA sequencingUsed to determine the bases along a

particular stretch of DNA

Was developed during the time of the Human Genome Project – allowed it to proceed much faster

What do you need?First we need:DNA of interest, with known sequences at the

endsDNA polymeraseATCG primers, modified A, T, C and G in each

test tube.

How does it work?1st. Double-stranded DNA is denatured2nd. Primers attach to known ends3rd. DNA polymerase adds nucleotides4th. All four nucleotides are mixed into test

tubes with the DNA of interest

continued5th. One of the four nucleotides mixtures has

an ‘A’ that has been modified – the ‘terminator’.

Then separate the pieces by running an them on an agarose gel (electrophoresis) – the place where an A (adenine) is will show up

The same happens for the other bases…..