English language

How to pronounce restriction fragment in English?

Full Transcript

hi everybody this is dr a and i have
your third video in molecular and
we are going to look at probe
hybridization techniques in this video
so a nucleic acid probe is a short
strand of dna or rna of a known sequence
that is well characterized so it's a
sequence for a gene that we're looking
for
and it is complementary for the base
sequence on the test target and that's
the the sequence we're trying to detect
the probes can be fragments of genomic
nucleic acids that can be made from
clone dna or rna or from synthetic dna
in a hybridization reaction the probe is
what is detected
and the way we detect it then is then we
label the probe
and so it can be labeled directly with a
radionuclide an enzyme or biotin
and the enzyme has to be able to
generate a colored reaction of some sort
or a fluorescent reaction or a
chemiluminescent reaction and then
that's what's detected
um biotin binds to avidin and then
avidin is usually bound to an enzyme and
then the enzyme is should be able to
generate a color reaction a fluorescent
reaction or a chemiluminescent reaction
hybridization of the probe to the target
dna or rna can take place in a solid
support medium or
in a solution
so this illustrates it so we would have
dna in the sample
we would denature it so that it becomes
single-stranded dna and then we would
incubate it with labeled dna probes that
are looking for a gene of interest which
is all purple here and
if the
you know the gene is there the probes
are complementary to that gene it will
anneal
and
then everything else that's not
hybridized or uh you know on a deal
there will
uh be washed off and then
the probes are detected presence or
absent and stuff so that is the basic
hybridization idea
so um why do we use hybridization
uh and what's the application of
hybridization probes so we can use them
to detect infectious organisms
to detect gene rearrangements and
chromosomal translocations and breakage
this can be helpful especially with
cancers
uh but also with genetic diseases we can
detect changes in occur genes and tumor
suppressor genes or factors we can aid
in prenatal diagnosis of an inherited
disease or detect a carrier status
we can use it to identify polymorphic
markers used to establish identity or
non-identity so that would be like
forensics paternity that kind of stuff
and it could also aid in donor selection
for um you know organ implants and stuff
transplants
so this is what a translocation is in
case you don't know so you would have
two um
basically chunks of
two different chromosomes that swap
uh
location
and so that is a translocation and that
happens a lot in cancers and stuff and
then uh this is an example of the
detection for of a cancer gene a
proto-auto gene
and so we're going to look at some of
these and how we can do those okay
so the solid support hybridization um we
have first a dot blot hybridization it
is the simplest type of solid support
hybridization
the clinical sample is applied directly
onto a membrane surface
the membrane then is heated which
separates the dna strand so that's the
denaturation step
then the label probes are added and then
a wash step removes any of the
unhybridized probes and then the
presence of the probes is detected
either via the audio
radiography or
the action of the enzymes producing a
color change or
a fluorescent light or something like
that
uh this one is a qualitative test only
so you only get
positive or negative it's there or it's
not all right
and it is subject to background
interference which makes weak positives
hard to interpret
so here it is illustrated so you have
your sample dna is extracted it is
applied to
the not on paper so it's blotted on it
not on paper in these little circles
it is denatured by alkali
and then
you have you add all the probes
that are labeled so you get probe
hybridization and uh it will hybridize
to the dots that
have the gene of interest the washtech
removes any of the unhybridized probes
and then uh the
autoradiography in this case on x-ray
film can detect
where the label probes are
next you have sandwich hybridization so
it is a modified blot to resolve the
background problems it uses two probes
one is mounted a membrane to capture the
dna and the other anneals to a different
site than the first probe and it is
labeled so that is what is detected
this means that the sample dna is
sandwiched between the capture probe and
the signal probe
but if you understand the concept of a
sandwich normally sandwiches like this
like bread bread you know meat in the
middle right well in this one it's
actually more of an open face sandwich
you're going to see here i've got an
illustration
this method does increase specificity uh
and it often uses microtiter plates
instead of membranes therefore it's more
easily automated
so
here it is we have the capture probe so
it can be on a well and then the analyte
of interest the gene sequence of
interest we're trying to detect on the
dna
and we incubate them together
the um if it's there and complementary
to the capture probe then it would
anneal to it and hang in there and then
we add the detection probe and if the
detection probe is complementary to the
rest of the sequence here then it can go
and anneal on that spot the spot that's
open
and then uh the detection
everything while everything does
unhybridize would be washed and then
this part of the molecule
that is attached to the probe can you
know be detected via whatever you know
type of molecule it was
um and so you can see how with the probe
with the capture probe and the detection
probe being kind of side by side it's
open face sandwich and it's capturing uh
the analog here that's going to be
laying on top of
the capture and detection detect
probe
so next we have southern blot it is
called southern blood because it was
developed by en southern and then
as we found different applications they
decided to go with the you know names of
the cardinal points so then we have
southern we have northern there's
western
blot and so
it is named after somebody that
initially so the
first step in southern blot is a dna
extraction from a sample using a
phenolic reagent then the dna is going
to be digested by a restriction enzyme
or restriction endonuclease
this will produce dna fragments
the dna fragments are then separated by
agarose gel electrophoresis
and then
those dna fragments are denatured so
become single stranded and transferred
into a natural cellulose membrane
by capillary action of salt solution or
by an electric current to transfer the
dna to that membrane
once the dna has been transferred onto
the membrane the label probe is added
the probe will anneal to its
complementary base sequence this form
visible bands on the membrane
the northern blot is the same process
but it is used for rna
and so here it is
illustrated so you've got
your sample with that's digested with
the restriction enzymes it is loaded in
the electrophoresis gel
electrophoresis is applied the
sample migrates into these bands you
know with fragments precipitating out at
different locations then it is
transferred this one is a capillary
reaction transferred
um to the nitrocellulose membrane and
then
once it's transferred to the natural
cellulose membrane it's incubated with
radioactive probes
the probes will anneal
to their complementary sequences
wherever they find them and that will
cause bands to be visible after
the paper was exposed to x-ray
film and then you can see the bands on
the x-ray film
so the restriction fragment length
polymorphism
is kind of application of that but it
helps evaluate the differences in
genomic sequence to identify individuals
in forensic or paternity testing
or to identify a gene associated with
the disease so you get dna extraction in
purification it usually comes from
leukocytes
possibly followed by pcr to amplify the
amount of dna that's present
and then you incubate it with
endonucleases
a specific restriction enzyme and what
it does is it will produce dna fragments
of different lengths that are unique to
individuals so where it cuts the
patterns of the wear it can cut is
unique
and so everybody gets a different
pattern of
fragment lengths
and then you apply the southern blot
technique which we just explained and
then this is
similar to what you would see after you
know the x-ray
process has been done
so next we're going to talk about the
solution hybridization so the principle
is that first
both target nucleic acids and probes
have to be free to interact in a
reaction mixture
uh this results in an increased
sensitivity compared to the solid
support hybridization
it requires a smaller amount of sample
and then second the other requirement of
solution hybridization is the probe
must be single stranded and must be
incapable of self-annealing so the
probes can't bind to each other anneal
to each other
so this is an example of institute
hybridization a type of solution
hybridization
first you have the nuclease
hybridization assay also known as the s1
nucleus cutting assay
so the ugly
oligonucleotide analyte is captured onto
the solid support such as a 96 well
plate by a capture probe so it
captures it
then uh it is incubated with s1 nuclease
so s1 nuclease will degrade any single
stranded dna or rna into oligo or
mononucleotides
and it leaves all the double stranded
dna or rna intact so anything that got
captured that has become double-stranded
in is
left alone right
then the double-stranded dna or
double-stranded rna then can be
precipitated and detected
the hybridization protection assay or
hpa the probe is labeled with acridium
ester which is chemiluminescent
then you have the hybridization step
and then you have an alkaline hydrolysis
to degrade the acrylium ester
of the unattached probes only it cannot
get to any probes that are already
attached and then
the probes that are
bound
then can emit light when the trigger is
added that trigger is hydrogen peroxide
the
dargine
hybrid capture 2 assay for the detection
of human papilloma virus is an example
of another solution hybridization
it uses an antibody that's specific to
the dna rna hybrids
to capture and detect them
the hybrids are made of one strand of
the hpv dna and a hybridized rna probe
and then that is what is detected by the
antibody
and then lastly in nc2 hybridization
this is a hybridization that is done on
cells tissues or chromosomes that have
been fixed on the microscope slide
it
uses heat denaturation of the slide to
separate the dna strands and then
incubation with the label probe to
anneal
uh and then the end product product will
generate either fluorescence or a color
change and then that's what's detected
so if you're looking for example these
are chromosomes if you're looking for
specific genes on a chromosome
everywhere where the probe has a neo
there is fluorescence and it can
then show where the location of that
gene or if that gene specifically is
present on those chromosomes this one is
more
on cells so anyway that wraps up our
hybridization video i have one more
molecular video coming up on dna
sequencing and
microarrays and stuff so stay tuned

hi everybody this is dr a and i have

Toggle Transcript

restriction fragment::noun | Spocien Dictionary

restriction fragment

noun

the fragment of DNA that is produced by cleaving DNA with a restriction enzyme

Type	Words
Type of	fragment

Examples of restriction fragment

restriction fragment

This became known as a restriction fragment length polymorphism, or RFLP.

From the genotopia.scienceblog.com

Measurement of telomere length by the Southern blot analysis of terminal restriction fragment lengths.

From the nature.com

On the average, microsatellite and restriction fragment length polymorphism loci yield identical estimates.

From the en.wikipedia.org

Restriction fragment length polymorphism analysis of the ribosomal DNA from the four species corroborated the results obtained from phylogenetic analysis.

From the en.wikipedia.org

Analysis of herpes simplex virus type 1 restriction fragment length polymorphism variants associated with herpes gladiatorum and Kaposi's varicelliform eruption in sumo wrestlers.

From the sciencedaily.com

The sickle cell mutation disrupts one of those restriction sites, so that the enzyme passes it over, making that fragment longer than normal.

From the genotopia.scienceblog.com

How to pronounce restriction fragment in English?

restriction fragment::noun | Spocien Dictionary

abandonment

Related words

Examples of restriction fragment

Related

Subscribe to our Newslatter