Transcript Electrophoresis and Blots

Electrophoresis and Blots
Principles recap
• Nucleic acids (DNA, RNA) move from the negative
to the positive pole during gel electrophoresis
• The resistance to movement is due to the sieving
effect of the gel matrix
• The rate of migration is related to the size of the
nucleic acid
Agarose versus acrylamide
• Acrylamide is a crosslinked synthetic polymer useful in
separating DNA molecules of a few hundred bases in length
– The crosslinking reaction is carried out within the molding
apparatus
– The final gel is transparent and usually used in a vertical apparatus
• Agarose is a polysaccharide similar to starch useful in
separating DNA and RNA molecules of a few hundred to
tens of thousands on bases long
– A gel is created by boiling agarose into solution and allowing it to
cool within a molding apparatus
– The final gel is translucent and usually used in a horizontal
apparatus
Restriction enzymes I
• Origin and native utility
– Some restriction enzymes are used by bacteria to
protect themselves from invading DNA
– A “restriction-modification” system exists that marks
bacterial DNA by methylating it on certain sequences
• A restriction enzyme specific for those sequences cannot digest
DNA if it is methylated
– Invading viral DNA is unmarked and subject to
cleavage at those sequences
• Restriction enzymes
– Generally bacterial in origin
Enzymes
• But may come from lower
eukaryotes, viruses or transposable
elements
– Cleave DNA at short palindrome
sites
• The palindromes may be 2 or more
nucleotides long
• Unusual forms may recognize
sequences up to 30 bases long
– These are used in site specific
recombination events
• Cleavage usually results in a 5’
phosphate and a 3’ hydroxyl
• The resulting ends may be single
stranded over a short region, or
double stranded (blunt ends)
• The cleavage sites may be
covalently linked back together by
DNA ligase
Restriction enzymes III
• A restriction enzyme with a 6 base recognition site
would produce on average a fragment size of 4096
bases in length when used to cut single copy DNA
– The average fragment size can be determined for any
recognition site by the formula 4n where n is the number
of bases in the recognition site
Size of recognition
sequence
4
5
6
8
Average size of
fragment
256
1024
4096
65536
Agarose gel
electrophoresis
• Gels are loaded with samples
of restricted DNA and
electrophoresed for minutes to
hours
• The voltage is turned off and
the gel is stained with the
intercalator ethidium bromide
– DNA may be visualized in the
gel by illuminating it with
ultraviolet light
– Linear relationship between
mobility and the logarithm of
the DNA size
Size determination
• Size markers are commonly
used in electrophoresis
experiments
• The distance migrated is
related to the inverse log of the
molecular weight of the DNA
fragment
Migration versus size
100000
Nucleotide base pairs
– These are duplex DNA
fragments of a known size
– The migration distance of a
fragment may be compared to the
migration of the size markers
directly on the gel
– Alternatively the migration of a
fragment may be determined by
interpolation from a plot of the
distance migrated by the size
markers
10000
1000
100
0
2
4
6
Distance migrated in cm
8
Digesting human
chromosomal DNA
• produces a range of fragment
sizes due to cleavage of single
copy DNA
– Three billion base pairs cleaved by
a restriction enzyme recognizing 6
bases results in 750,000 fragments
• They appear as a background
smear of DNA cleaved into all
possible sizes by the restriction
enzyme
• The fragments are of random size
with an average length of >4096
bases
– Repetitive sequence can increase
the average size, because usually
a given restriction site will not
be found within it
– If a restriction site is in a repetitive
sequence, digestion will produce a
large number of fragments of
identical length
• This will appear as a band in the
background smear of fragments
• Once an agarose gel of digested chromosomal DNA
has been stained, individual DNA sequences may be
detected on the gel by hybridization
Southern
Blot
– The DNA within the gel is denatured by exposing it to a
solution of sodium hydroxide
– The DNA is then neutralized and transferred out of the gel
onto a membrane that binds DNA
• This is a procedure known as blotting
• It exposes the DNA to the surface so that it may hybridize to
complementary sequences
– The membrane bound DNA is then hybridized to a short
specific sequence known as a probe
Probe
• A probe is an oligo or poly nucleotide that
represents known DNA sequence
– It is usually a sequence complementary to a
gene
• But it only need be complementary to DNA
sequence within the chromosomal DNA itself
– It is either single stranded to begin with or is
denatured to make it single stranded
– It is labeled with radioactive phosphorous or a
fluorescent adduct
Hybridization
• The membrane is
“prehybridized” with nonspecific DNA to block nonspecific binding sites on the
membrane prior to
hybridization
• It is then hybridized to the
probe
– The temperature and salt
conditions are controlled to insure
optimal hybridization between the
probe and the target sequences
– The single stranded probe finds
its complement in the membrane
bound DNA and forms a double
helix
• Unbound probe is then washed
off
X-ray film
exposure
• The membrane is exposed to
X-ray film
– Radioactive probes expose the
film whereever they hybridized
on the blot due to emission of
electrons (beta particles)
– Fluorescent probes catalyze a
light yielding reaction using
energy supplied in a reaction
cocktail
• Development of the X-ray
yields an autoradiogram
– Bands on the film locate the
chromosomal restriction
fragments complementary to the
probe DNA
• Southern Blots
– DNA is digested with restriction enzymes and
electrophoresed through an agarose gel
– The contents of the gel are denatured in situ
and then transferred onto a membrane that
binds DNA
– The DNA is hybridized to radioactive DNA
complementary to the gene of interest
– Unhybridized DNA is washed away and the
membrane exposed to X-ray film
• The technique can be adapted to identify
any source of DNA containing a gene of
interest or to RNA detection (northern
blots)
Overall
Restriction fragment length
polymorphism (RFLP)
• Chromosomal DNA from two people differs due
to a random variations in sequence
– There are millions of single nucleotide polymorphisms
between two unrelated individuals
• If one of these sequence variations results in the
creation or destruction of a restriction site, then
the fragment sizes of DNA will differ between
these two people
• This can be detected by southern blots
Example I RFLP point mutation
• A gene sequence contains two Eco R1 sites
separated by 4000 nucleotides
• A patient has suffered a mutation in one of his two
homologous chromosomes such that a single
nucleotide change has created a new EcoR1 site
within the gene sequence
– Now two fragments are created of 1500 and 2500 bases
in length
– The probe is complementary to sequence located within
the 2500 base long fragment
– Detection by southern blot of these two fragments will
result in a 4000 base long fragment in normal DNA
from one homologous chromosome and two fragments
from the other homolog
DNA from normal individual
digested with Eco R1 and
electrophoresed through agarose
DNA from mutant individual digested
with Eco R1 and electrophoresed
through agarose
Example II RFLP
deletion mutation
How could these two
patterns arise by deletion?
Example III
Amplification
Other blots Northern
• Northern blots are mainly used to measure of the
amount of a specific RNA in the cell
• The blot is prepared and hydridized like a
southern blot
• There are a few minor technical alterations
– The RNA is usually folded into a secondary structure so
it must be denatured before and during electrophoresis
• Bands on the autoradiogram are a measure of the
presence of RNA from a particular gene
– The more intense the band, the more RNA on the blot
Protein electrophoresis
• Proteins, unlike DNA, do not have a constant size
to charge ratio
– In an electric field, some will move to the positive and
some to the negative pole, and some will not move
because they are neutral
– Native proteins may be put into gel systems and
electrophoresed
• However native proteins are sometimes difficult to prepare
without degradation
– An alternative to native protein gels forces all proteins
to acquire the same size to charge ratio
SDS-polyacrylamide gel
electrophoresis
• A sample of protein, often freshly isolated and
unpurified, is boiled in the detergent sodium dodecyl
sulfate and beta-mercaptoethanol
– The mercaptoethanol reduces disulfide bonds
– The detergent disrupts secondary and tertiary structure
– On the molecular level, proteins are stretched out and
coated with the detergent (which has a negative charge)
by this treatment
– They will then migrate through a gel towards the positive
pole at a rate proportional to their linear size
• Molecular weights with respect to size markers may then be
determined
Other blots II
Western
• Western blots measure the amount of
a specific protein in a cell
• This is a blot of an SDSpolyacrylamide gel onto which
cellular protein is applied
– The protein is denatured so it will enter
the gel as a stretched out random coil
– The proteins separate by size
– They are blotted onto a membrane
• Western blots are probed using
antibodies
– The antibodies are labeled. Those that
remain bound to their specific targets
identify specific proteins in the cell
• The intensity of the signal reflects
the level of expression
• It can also reveal mutations by
anomalies of migration
– A deletion might cause a shorter protein
for example