PINES CITY COLLEGES

COLLEGE OF MEDICAL LABORATORY SCIENCE

2nd SEMESTER A.Y. 2022-2023
MOLECULAR BIOLOGY LEC

TITLE: BLOTTING TECHNIQUES

Southern Blots
 Named for Edwin Southern, who first reported the procedure.
 DNA is isolated and cut with restriction ____________.
 The fragments are separated by ____________________, ________________ and
____________, and then transferred to a solid support such as ______________.
 In the final steps of the procedure, the DNA fragments are exposed to a labeled probe
that is specific in sequence to the region of interest, _______________ is removed, and
the signal of the probe is detected to indicate the presence or absence of the sequence
in question.
 The original method entailed hybridization of a ______________ labeled probe to
detect the DNA region to be analyzed.

Restriction Enzyme Cutting and Resolution

 The first step in the Southern blot procedure is __________of the DNA with
restriction enzymes. The choice of enzymes used will depend on the applications.
 For routine laboratory tests, _________________ of the target DNA regions will have
previously been determined, and the appropriate enzymes will be recommended.
 ________ of genomic DNA are used for each restriction enzyme digestion for Southern
analysis. More or less DNA may be used depending on the sensitivity of the detection
system, the volume and configurations of wells, and the abundance of the target DNA
 After restriction enzyme digestion, the resulting fragments are resolved by
_________________________. The percentage and nature of the gel will depend on
the _____ of the DNA region to be analyzed
 A molecular weight standard should be run with the test samples. After electrophoresis,
it is important to observe the ____ DNA.
 Genomic DNA cut with restriction enzymes should produce a smear representing the
billions of fragments of all sizes released by the enzyme cutting. The brightness of the
DNA smears should be similar from lane to lane, assuring that equal amounts of DNA
were added to all lanes. In any lane, a large aggregate of DNA near the top of the gel
indicates that the restriction enzyme activity was ____________. A smear located
primarily in the lower region of the lane is a sign that the isolated DNA is
_____________. Either of these two latter conditions will prevent accurate analysis.

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Preparation of Resolved DNA
 The goal of the Southern blot procedure is to analyze a ________________ of the
sample DNA.
 The resultant restriction fragments containing the target sequence to be analyzed are
obviously not distinguishable in the smear from other fragments that do not have the
target sequence.
 Target fragments can be detected by _______________ with a homologous sequence of
single-stranded DNA or RNA labeled with a detectable marker.
 To achieve optimal hydrogen bonding between the probe and its complementary sequence
in the resolved sample DNA, the double stranded DNA fragments in the gel must be
___________ and _____________ to a nitrocellulose membrane.

Depurination
 Before moving the DNA fragments from the gel to the membrane for blotting, the
double-stranded DNA fragments must be denatured, or separated, into single strands.
This is performed as the DNA remains in place in the gel. Although short fragments can
be denatured directly, larger fragments are more efficiently denatured if they are
depurinated before denaturation . Therefore, for large fragments, the gel is first soaked
in ____solution, a process that removes _________ bases from the sugar phosphate
backbone. This will “loosen up” the larger fragments for more complete denaturation.

Denaturation
 The DNA is denatured by exposing the DNA in the gel to _______________. The strong
base NaOH promotes breakage of the _______________ holding the DNA strands to
one another.
 The single-stranded DNA will bind more tightly than double-stranded DNA to the
nitrocellulose membrane upon transfer.

Blotting (Transfer)
 Before exposing the denatured sample DNA to the probe, the DNA must be transferred,
or blotted, to a solid substrate that will facilitate probe binding and signal detection.
This substrate is usually ______________, ______________________,
____________________ modified with a diethyl amino ethyl, or a carboxy methyl (CM)
chemical group.
 _______________________), are used for immobilizing proteins for probing with
antibodies (Western blots).

Transfer Methods
 The original method developed by Southern used ________________ .

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  the gel is placed on top of a reservoir of buffer, which can be a shallow container
or membrane papers soaked in high salt buffer, e.g., 10X saline sodium citrate (10X
SSC: 1.5 M NaCl, 0.15 M Na citrate) or commercially available transfer buffers.
The nitrocellulose membrane is placed directly on the gel, and dry absorbent
membranes or paper towels are stacked on top of the membrane. The buffer is
moved by capillary action from the lower reservoir to the dry material on top of
the gel. The movement of the buffer through the gel will carry the denatured DNA
out of the gel. When the DNA contacts the nitrocellulose membrane, the DNA will
bind to it while the buffer will pass through to the membranes or paper towels on
top.
 ______________ transfer, uses electric current to move the DNA from the gel to the
membrane .
 This system utilizes electrodes attached to membranes above (anode) and below
(cathode) the gel. The current carries the DNA transversely from the gel to the
membrane. Electrophoretic transfer is carried out with a “tank” or by a “semidry”
approach.
 __________________ the electrodes transfer current through the membrane through
electrophoresis buffer
 __________________the electrodes contact the gel-membrane sandwich directly,
requiring only enough buffer to soak the gel and membrane.
 The tank electrophoretic transfer is preferred for _______________ resolved on
acrylamide gels, whereas the semidry method is frequently used for __________
proteins.
 ________________ is a third method of DNA blotting
 This blotting technique uses suction to move the DNA from the gel to the
membrane in a recirculating
buffer. Like electrophoretic transfer, this method transfers the DNA more rapidly
 discontinuous transfer due to air trapped between the membrane and the gel is
avoided.
 One disadvantage of the second and third methods is the expense and maintenance
of the electrophoresis and vacuum equipment.
 After binding the nucleic acid to membranes, the cut, denatured DNA is permanently
immobilized to the membrane by baking the membrane in a vacuum oven (80C, 30–60 min.)
or by UV cross-linking, i.e., covalently
attaching the DNA to the nitrocellulose using UV light energy.
 The purpose of baking or cross-linking is to prevent the transferred DNA fragments
from washing away or moving on the membrane.
 A ______________________ step is required to prevent the probe from binding to
nonspecific sites on the membrane surface, which will cause high background.

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  Prehybridization involves incubating the membrane in the same buffer in which the
probe will subsequently be introduced.
 The buffer consists of blocking agents such as Denhardt solution (Ficoll, polyvinyl
pyrrolidane, bovine serum albumin) and salmon sperm DNA.
 Sodium dodecyl sulfate (SDS, 0.01%) may also be included, along with formamide
 The membrane is exposed to the prehybridization buffer at the optimal
hybridization temperature for 30 minutes to several hours, depending on the
specific protocol.
 At this stage the sample is ready for hybridization with the probe, which will allow
visualization of the specific gene or region of interest.

Northern Blots
 Designed to investigate _______________________.
 the method can also be used to investigate RNA structural abnormalities resulting from
aberrations in synthesis or processing, such as _______________.
 Splicing abnormalities are responsible for a number of diseases, such as
_________________ and __________________________________.
 Analysis of RNA structure and quantity indirectly reveals mutations in the regulatory or
splicing signals in DNA.
 After isolation and quantitation of RNA, the samples (up to approximately 30 g total RNA or 0.5–
3.0 g polyA RNA, depending on the relative abundance of the transcript under study) can be
applied directly to agarose gels.
 ________ concentrations of 0.8%-1.5% are usually employed.
 _______________________ can also be used, especially for smaller transcripts
 Gel electrophoresis of RNA must be carried out under denaturing conditions for accurate
transcript size assessment.
 ___________________ is also required for efficient transfer of the RNA from the gel to the
membrane, as with the transfer of DNA in the Southern blot.
 Representative lanes can be cut from the gel, soaked in ________________ to remove the
denaturant, and stained with acridine orange or ethidium bromide to assess quality and equivalent
sample loading.
 Denaturant, such as ____________, must be removed from the gel before transfer because it
inhibits binding of
the RNA to nitrocellulose.
 This is accomplished by rinsing the gel in ___________ water.
 RNA is transferred in 10X or 20X SSC or 10X SSPE (1.8 M NaCl, 0.1 M sodium phosphate, pH 7.7,
10 mM EDTA) to nitrocellulose as described above for DNA. 20X SSC should be used for
small transcripts.
 The blotting procedure for RNA in the Northern blot is carried out in 20X SSC, similar to the
procedure for DNA transfer in the Southern blot.

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  Prehybridization and hybridization in formamide/SSC/SDS prehybridization/hybridization
buffers are performed also as with Southern blot. If the RNA has been denatured in glyoxal, the
membrane must be soaked in warm Tris buffer (65C) to remove the denaturant immediately
before prehybridization.

Western Blots
 The immobilized target for a Western blot is ___________.
 serum, cell lysate, or extract is separated on SDS-polyacrylamide gels (SDS-PAGE) or isoelectric
focusing gels (IEF).
 The former resolves proteins according to ______________, and the latter according to
charge.
 __________________________ can also be used to separate proteins into subunits.
 Polyacrylamide concentrations vary 5%-20%.
 Depending on the complexity of the protein and the quantity of the target protein, 1–50 g
of protein is loaded per well.
 Before loading, the sample is treated with denaturant, such as mixing 1:1 with 0.04 M Tris
HCl, pH 6.8, 0.1% SDS.
 The accuracy and sensitivity of the separation can be enhanced by using a combination of
IEF gels followed by SDS-PAGE or by using two-dimensional gel electrophoresis.
 Prestained molecular weight standards are run with the samples to orient the membrane
after transfer and to approximate the sizes of the proteins after probing.
 The gel system used may affect subsequent probing of proteins with antibodies.
Specifically, denaturing gels could affect epitopes such that they will not bind with the
labeled antibodies.
 Gel pretreatment with mild buffers such as 20% glycerol in 50 mM Tris-HCl, pH 7.4, can
renature proteins before transfer.
 Proteins can be blotted to membranes by ___________________________ transfer.
 ________________ has high affinity for proteins and is easily treated with detergent (0.1%
Tween 20 in 0.05 M Tris and 0.15 M sodium chloride, pH 7.6) to prevent ____________ of the
primary antibody to the membrane itself before hybridization.
 Binding of proteins to nitrocellulose is probably ________________ as nonionic detergents can
remove proteins from the membrane.

Education is the passport to the future, for tomorrow belongs to those who prepare for it
today.
-Malcolm X-

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