Honors Biology 298 - Week 12
Once a cloned sequence representing all or a portion of a gene has been identified and selected from a library, it can be used to for many things like
1) DNA sequencing –
most sequencing today is fully automated
ex: used in human genome project
2) polymerase chain reaction (PCR)
much like cloning, but even faster and more efficient
PCR uses single-stranded DNA as a template for the synthesis of a complementary
strand
In this reaction:
the DNA to be amplified is heated
short nucleotide sequences (usually 20-30 nucleotides long) that act as primers for DNA replication are added
DNA polymerase begins at the primers
this series of three steps constitutes
the entire cycle can be repeated numerous times
the end result is that the amount of DNA present doubles with each cycle
several advantages to this technique
DNA to be amplified does not have to be purified
DNA from many sources can be used
ex:
PCR is also used in clinical diagnosis, forensic application
how are the fragments generated by cutting with restriction enzymes separated?
run what is called
in this technique, genomic DNA is cut into fragments
the DNA fragments are then separated by gel electrophoresis
as fragments move through the gel, they separate by size
then stain gel with a DNA fluorescent dye and expose to UV light
want to then use a probe and identify a complementary sequence within genomic DNA
denature DNA in gel (single-stranded fragments) and transfer them to a sheet of
the transfer is accomplished by placing the membrane on top of the gel and
allowing a buffer solution to flow by capillary action
in this manner, movement of the buffer transfers DNA fragments
this membrane is then exposed to a radioactively-labeled probe
in order to visualize hybridized probe/DNA fragments, a piece of X-ray film is
placed over the membrane
3) mapping a gene at the chromosomal level
if a cloned gene has been recovered from a library
this cloned gene is labeled
in the technique known as fluorescence in situ hybridization (FISH)
the probe forms a hybrid with complementary sequences on the chromosome and
when viewed under a microscope, the location is marked by the presence of a
bright fluorescent spot
has helped in identifying the locations of many genes
DNA Fingerprinting
a very widely publicized technique
based on the use of restriction enzymes
more specifically, it detects variations in the number of short, repeated DNA sequences
these sequences are called minisatellites
ex:
this sequence is made up of 6 tandem copies of a 5-nucleotide sequence CCTTC
there are many different types of repeats
clusters of such repeats are scattered
furthermore, the number of repeats at each site is variable, ranging from 2 >100
because everyone’s DNA is different, the number of these tandem repeats varies
as a result, the pattern of DNA fragments produced when a person’s genomic DNA is cut by restriction enzymes is unique
by definition, DNA fingerprinting is the pattern of restriction fragments
in criminal cases, DNA is extracted from biological material left at a crime scene
this DNA is cut with restriction enzymes
if you were a juror sitting on one of these cases, what questions would be raised in your mind on the validity of this procedure? (do you see any potential problems with this technology?)
to overcome some of the potential artifactual results
DNA fingerprinting is not done on one sample
instead, individual probes are used to determine a fingerprint pattern
in most cases, at least 4 different probes are used
if the DNA fingerprint profile of an individual does not match that of the evidence
if the evidence and individual’s profiles match
O.J. Simpson’s trial: probability (frequency of these tandem repeats in black males)
so they computed the frequency that a particular pattern of tandem repeats will be present
even though individual frequencies might be fairly common, the combination of
frequencies usually results in a rare event
DNA fingerprints have been used in a variety of applications
the most widely used application of fingerprinting is
furthermore, using recombinant DNA techniques, it is now possible to do both
prenatal testing
used in conjunction with amniocentesis or chorionic villus sampling
can you think of one genetic disorder that we have talked extensively about that
cannnot be prenatally diagnosed using conventional methods but would make a
good candidate for recombinant technology?
this condition results from a simple mutation
this changes the length and number of DNA fragments generated
can screen parents for this mutation
another type of prenatal testing:
called preimplantation testing
human eggs are fertilized in vitro
then one cell of an 8-cell stage embryo is removed and the DNA is extracted
ex: cystic fibrosis, muscular dystrophy, hemophila
embryos lacking any genetic disorder are then implanted
presymptomatic testing is genetic testing for adult-onset disorders where testing can be done at any time before symptoms occur (prenatal through adult)
detection of disorders that occur later in life
symptoms arise between 35-50 yrs of age