Recombinant DNA Technology
Making of a clone:
what is a clone?
what can be cloned?
this technology has had a great impact on genetic research and has generated
applications of this technology across many disciplines
recent advances in the cloning of plants and animals
domesticated plants and animals have been genetically manipulated
ex: cows, sheep, dogs
those organisms with desirable characteristics were selected and then bred
this slow procedure can be used to produce desirable individuals
plants
back in the 50’s, it was demonstrated that individual carrot cells could be removed from a plant and when grown in culture under the appropriate conditions
these calluses could then be stimulated to form roots and shoots
can you see the benefit of this?
what types of characteristics would be desirable?
from a few cells, upwards of thousands of copies of the original tree could be produced
animals
cloning of domesticated animals like cattle and sheep
in cloning of animal embryos, there are 2 ways to accomplish this
1) embryo splitting –
the embryo is then physically divided 1 or more times
individual cells (genetically identical) can then be implanted
this method mimics the way identical twins (triplets) are produced
2) nuclear transfer –
in this method, eggs are removed from a donor animal
these enucleated eggs are then injected with pluripotent nuclei
if this transfer is successful
when these cells develop to the 16-32 cell stage
all these embryos can be transferred to surrogate females
commercialization of this technique
farmers might become geneticists/molecular biologists and use a kit to
create a “superherd”
ex: cloning of the sheep named Dolly
Scottish scientists produced a clone of a sheep using a nucleus from a
differentiated cell
a mammary cell with an intact nucleus was taken from the udder of one
sheep and fused with the enucleated egg from another sheep
this fusion product was then implanted into a surrogate female sheep
the result was a new sheep that was an exact copy or clone of the sheep
so how efficient or successful is this technique?
in the case of Dolly, it took
but it does give the potential for the cloning of animals
what does this prove about differentiated cells?
why so many attempts to finally be successful?
we now have at our disposal known methods for cloning plants and animals, where do we go from here?
do you think it is possible to use such techniques to create new life forms
these techniques present us with the possiblity of even transferring genes
ex: plants have some type of defense mechanism against a set of carcinogenic
substances that is controlled by one or a few genes……we don’t have this sytem
the biotechnology of genetic engineering and cloning is now crossing over into conservation biology as we struggle to maintain or conserve biological diversity in all
different types of animals including endangered species
many zoos have instituted captive breeding programs that use such technology and now have methods for embryo transfer where embryos from rare and endangered species can be implanted in common, closely related, surrogate host species and carried to term
ex: Cincinnati Zoo has been using embryo transfer technology since 1984
could we then generate a situation like Jurrasic Park?
Cloning Genes is a Multistep Process
cloning DNA is similar to cloning whole plants or animals
genetic and biochemical discoveries were made back in the early 70’s
the basics include:
1. produce DNA fragments using enzymes
2. link these fragments to self-replicating forms of DNA
3. replicate these recombinant DNA molecules
4. retrieve the cloned DNA insert
5. produce and purify expressed gene products
what are restriction enzymes?
first identified in soil bacteria
definition: enzymes that recognize a specific base sequence and attach to the DNA
molecule
the recognition sequence is usually a 4-6 base pair palindrome
restriction enzymes are named for the organisms from which they are isolated
recombinant DNA molecules are created using
why use 1 enzyme to cut each source of DNA?
restriction enzymes allow DNA fragments from different organisms to be joined together
have a whole series of these enzymes
what are vectors?
vectors are used to carry DNA segments
many of the vectors used in recombinant DNA work are derived from
plasmids are extrachromosomal DNA
most plasmids can carry up to several thousand base-pairs of DNA
plasmids are modified to contain many restriction sites ex: pBR322
also have what are called YACs
contains 2 telomeres, a centromere and a region with a cluster of restriction enzyme sites
the recombinant artificial chromosome is transferred to a yeast cell
this type of vector has been very important in the human genome project
complete process of cloning DNA
most bacteria are naturally competent –
if not, then some sort of treatment is necessary like exposing the bacteria to a salt
solution and applying a heat shock
transformation can also be accomplished by
these transformed bacterial cells are grown on a nutrient plate
now need to identify which colonies (and the cells within) have taken up recombinant plasmids
do this by differential screening of colonies
replica plating
can the entire genome of an organism be cloned?
how would this be done?
because each cloned fragment of DNA is small, relative to the size of the genome, many separate clones must be created
a collection of clones that contains all the DNA sequences in the genome is called a
there is a particular gene I am interested in, how do I go about locating it in a library?
using other techniques of molecular biology, any sequence or gene of interest can be recovered from such a library
most often, a specific clone is identified using a radioactively labeled nucleic acid
probes can be radioactive DNA or RNA molecules
can also use other methods that use chemical reactions or color reactions
the ability of a probe to recognize a piece of DNA (clone) in a library depends on the fact that under the right conditions, 2 single-stranded nucleic acid molecules with complementary base sequence will form a double-stranded hybrid molecule
because the probe is labeled in some way, the clone carrying a DNA insert that hybridizes with the probe can be identified
probes can come from a variety of sources
ex:
why was this possible?
ex: can use cDNA
what about creation of a cDNA library?