0:00
dna extraction and preparation is the
0:02
crucial first step in the southern
0:04
blotting technique this process begins
0:07
with extracting highquality DNA from
0:09
biological samples such as blood tissue
0:11
or cultured cells the extraction process
0:14
involves several critical steps to
0:16
isolate DNA while preventing
0:18
degradation first cells are liced to
0:21
release their contents then proteins
0:23
lipids and other cellular components are
0:26
removed through chemical and physical
0:27
treatments after initial extraction the
0:30
DNA must be purified to remove any
0:33
remaining proteins RNA and chemical
0:35
contaminants that might interfere with
0:37
downstream applications for southern
0:39
blotting 5 to 10 micrograms of
0:41
highquality DNA is typically required
0:44
the purity is often assessed using
0:46
spectrophotometry with an A260 to A280
0:49
ratio of approximately 1.8 indicating
0:52
DNA free from protein
0:54
contamination once highquality DNA has
0:57
been extracted and purified it is ready
0:59
for the next step in southern blotting
1:02
restriction digestion after probe
1:04
hybridization the southern blot membrane
1:07
underos a series of washing steps the
1:09
washing process serves three main
1:11
purposes to remove unbound probes reduce
1:14
background signal and control the
1:16
specificity of hybridization the
1:18
membrane contains both specifically
1:20
bound probes that have hybridized to our
1:22
target sequences and unbound probes that
1:25
bind non-specifically or remain in
1:28
solution a series of buffer washes
1:30
removes these unbound probes leaving
1:32
only the specifically bound probes
1:34
behind the stringency of these washes
1:37
can be controlled through salt
1:38
concentration and temperature
1:41
higher stringency washes provide greater
1:43
specificity but may reduce sensitivity
1:46
while lower stringency washes do the
1:48
opposite after washing the membrane is
1:51
ready for detection the method used
1:53
depends on the type of probe label
1:55
radioactive probes are detected by
1:58
exposure to X-ray film or using
2:00
phosphor fluorescent probes require
2:03
optical imaging systems while
2:05
chemoluminescent probes create light
2:07
through enzyatic reactions the detection
2:09
process reveals a pattern of bands
2:11
representing the target DNA fragments
2:14
these bands indicate the presence of
2:17
your target sequence their positions
2:19
show the fragment sizes and the
2:21
intensity of each band correlates with
2:24
the abundance of that fragment with
2:25
detection complete we now have valuable
2:28
data about our DNA sample that can be
2:30
interpreted and applied in various
2:32
molecular biology applications in the
2:35
next section we'll explore the
2:36
applications of southern blotting in
2:39
diagnostics southern blotting has
2:41
diverse applications in molecular
2:43
biology and genetics let's explore the
2:46
five major applications where southern
2:48
blotting remains a valuable
2:50
technique gene mapping is a crucial
2:53
application where southern blotting
2:55
helps scientists locate specific genes
2:59
dna fingerprinting is another key
3:01
application used in forensics and
3:03
paternity testing where southern
3:05
blotting reveals unique genetic patterns
3:09
individuals southern blotting is
3:11
critical in diagnosing genetic disorders
3:14
by detecting mutations in DNA such as
3:16
insertions deletions or structural
3:19
changes in cancer research southern
3:22
blotting helps scientists detect gene
3:24
amplifications or deletions that can
3:27
contribute to tumor development and
3:30
progression southern blotting is used to
3:33
verify genetically modified organisms by
3:35
detecting the presence of inserted
3:37
foreign DNA sequences in the genome
3:40
despite the emergence of newer
3:42
technologies southern blotting remains
3:44
valuable for specific applications where
3:46
high sensitivity and specificity are
