DNA Hybridization and Blotting techniques

Hybridization

● is the double stranded formation or annealing between two different single strand DNA or RNA (either of the two)
○ Due to complementary base pairing region of this two nucleic acids

■ Not typical double bonded formation; let say between the DNA double helix kase yung isang DNA is the DNA from the genome
■ And the other DNA which will bind to the specific DNA in the genome is LABORATORY MADE DNA.

○ Hybridization may also happen between DNA and RNA especially if you are looking for a specific gene or specific mRNA transcript - so you can also use DNA that will bind to that RNA -another hybrid may form (combination of DNA and RNA)

Laboratory made DNA

■ has it specificity to the region of the entire

DNA

RNA probes

● are possible which can detect specific RNA transcript and form hybrid to that transcript
○ this phenomenon forms the basis of several nucleic acid techniques

■ includes some steps in southern blotting technique, FISH (fluorescence in situ hybridization) technique
■ along with the hybridization technique is the blotting technique

● pwedeng combination tung dalawang technique na to (hybridization and blotting techniques)
● these two are different techniques but you can still combine them in one particular method
○ ex. in southern blotting technique - there is blotting technique, blotting steps, as well as hybridization steps

○ that is why it is important to understand the principle behind this type of techniques particularly DNA hybridization and blotting techniques

Plot analysis technique

● is a powerful method to detects a specific biomolecule such as nucleic acids as well as proteins in sample of complex composition

PROBES

In mol bio,when we performed techniques to study nucleic acid and other important biomolecules - we usually use we called

○ Probes

■ is the stretches of nucleic acid

■ mga oligonucleotides

■ short stretches of DNA or it may also RNA that where the labeled attach to
■ DNA and RNA probes are short segments of nucleic acids of known base sequences
■ it is used to look for specific regions in the DNA of sample or a cell - that compliments to the base sequence of the probes
■ usually these probes are labeled with either radio isotope or fluorescent material- so that we can follow where is the attachment of the DNA probes

○ Label

■ allow us to see the where DNA binds either on the cell or on chromosome or even in the pure isolated DNA
■ usually we labeled the probes with different molecules to follow them
■ we can use radioactive material sa mga isotopes or fluorescent material to chemically attach into a probes

DNA probes

● is use in hybridization techniques

Hybridization

■ is fundamental property

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■ basic property of double stranded DNA molecules due to complementary base pairing
■ this property is advantageous for detecting the presence of specific DNA or RNA in a mixture
■ also can be used for estimating the quantity of the NA

Hybridization

observed between (a) a DNA probe and a DNA target and (b) a DNA probe and an RNA target

● This is DNA probe
● which is a short segment of DNA
● normally it is labeled with the fluorescence or radioactive material
● this short oligonucleotides has specific base sequence; synthesize in the laboratory
○ and this base sequence is complementary to the gene what we are looking for to the gene of interest

Hybrids

DNA probes + gene of interest =

hybrids

there would be a complementary base pairing between the two and therefore, will forms

Southern blotting

● is performed in order to detect the presence of specific DNA

heteroduplex

DNA probe with base sequence complementary to the mRNA sequences; due to complementary base pairing magkakaroon ng DNA and RNA hybrids also called as

blotting techniques

● which can be found as one of the steps in several molecular assay including southern blotting, northern blotting and western blotting

● NA/proteins is immobilized (they are fixed) on a solid support (e.g. nylon or nitrocellulose membrane)

The NA acid

will be fixed onto that nylon membrane from a gel electrophoresis

○ Southern blot

■ in which in both probe and target nucleic acid are DNA

■ use to detect the presence of specific DNA by blotting techniques with the use of DNA probes

○ Northern blot

■ in which the probe is a single stranded DNA

■ whereas the target is usually mRNA

○ Western blot

■ USE to detect the presence of proteins in the sample

SOUTHERN BLOT

● It refers to the technique of transferring DNA fragments from electrophoresis gel to membrane support and subsequent analysis of the fragments by hybridization with radioactive or fluorescently labeled DNA probes
● Southern blotting is named after Edwin M. Southern, who in 1975 first described the technique of DNA analysis
● It enables reliable and efficient analysis of DNA fragments in immobilized membrane support such as nitrocellulose membrane.

HOW TO CUT DNA INTO SHORT FRAGMENTS?

i. using restriction enzymes,
ii. restriction endonuclease - are use to cut the high molecular weight DNA strands into smaller fragments
b. so when we obtain complete dna fragments, we can now performed southern blot analysis
c. reminder: you have to use a short fragments of DNA

2. The DNA fragments are separated using gel electrophoresis

a. gel electrophoresis - separates the fragments according to their size
b. fermented DNA typically electrophoresed on an agarose gel - to separate the fragments according their molecular weights

3. We will denatured the DNA - because DNA is double stranded - we need to separate the DNA

a. WHY DO WE NEED TO SEPARATE DNA?

i. since we have to bind the DNA probe here to the DNA INTEREST

ii. PAANO MAG BIBIND YUNG DNA PROBE DOON SA DNA INTEREST IF IF ITS DOUBLE STRANDED? KAYA NEED MAG DENATURED SI DNA
b. DENATURATION STEP:

i. The DNA gel electrophoresis is soak in alkaline solution (typically sodium hydroxide)- to separate the DNA double strands

● this is required for hybridization later, kapag mag bibind na yung DNA probe
● dapat single stranded and DNA of interest

● The alkaline solution also destroys any residual RNA that may be still present in the DNA

4. Blotting technique

a. The DNA from the gel will be transferred onto nitrocellulose membrane
b. a sheet of nitrocellulose or nylon(alternatively use) is placed on top or below of the gel (depending on the direction of the transfer)

i. it is actually either top or below depending on the direction of the transfer kung pataas o pababa
ii. pataas ang transfer dapat yung nitrocellulose or nylon membrane ay nasa ibabaw ng gel
iii. direction of transfer or movement ng DNA ay pababa DAPAT YUNG MEMBRANE
c. Buffer

i. usually the use buffer here is saline sodium citrate (SSC)

BUFFER

ii. The buffer is drawn up on the top layer of the blotting paper passes through the gel
iii. As the buffer passes through the gel it carries the DNA

● gel is a porous membrane (butas butas), it has microscopic pores on the gel.
a. so it allowed the passage of molecules as well as the water
b. SO it is POSSIBLE FOR DNA TO LEAVE THE GEL and for WATER to passed through the gel
iv. The DNA ay pumupunta ngayon sa membrane, it attached itself to the membrane
v. The buffer is drawn up due to capillary reaction - like the water in the buffer; the buffer is pataas sya through capillary action - it defies gravity

● habang pataas yung water from the gel; it starts at the bottom as nandon yung buffer
● tataas yung buffer, sisipsipin sya ng sponge
● The sponge (in the pic above) are soak with buffer (saline sodium citrate buffer)
● On top of the sponge is whatman paper yung filter paper
● on top of filter paper is a gel that containing the DNA
● on top of gel electrophoresis is the nitrocellulose or nylon membrane
● at the top of, has a several layer of whatman paper, then paper towel, then at the top is weight (para equal yung distribution ng pressure)
● The buffer/water goes up through capillary action (pataas ang movement ng buffer)

ion-exchange interaction

● Another driving force that transfer DNA from the gel to the membrane is the ___________________ between the DNA and nitrocellulose membrane

5. Immobilization step

a. is also known as the fixation step,
b. in this step , ifi-fix, ididikit yung DNA sa nitrocellulose membrane
c. The nitrocellulose or nylon membrane is baked (put on inside the irregular oven) at 70 - 80°C for 2 hrs.
i. in this way mafifix yung DNA sa nitrocellulose membrane
d. Alternatively: we can use ULTRAVIOLET RADIATION especially when you are using Nylon membrane
i. Heat is 70 - 80°C - applicable for both membrane
ii. But Ultraviolet radiation is used for immobilization ONLY IF USING NYLON MEMBRANE
iii. This will permanently attached or immobilized the transfer of a membrane
iv. After immobilization step, the transfer the DNA in the membrane is now fixed

6. HYBRIDIZATION STEP

. The membrane is now exposed to the DNA probe

i. RECALL: DNA probe -is a single fragment with a specific sequence who is present in the target DNA to be determine

ii. The probe DNA is labeled so as we can detected; by incorporating radioisotopes, tagging the molecule with fluorescence dye

iii. In some cases hybridization probe or dna probe are NA acid probe that may be made from RNA rather than DNA
● pwedeng RNA probe or DNA probe

iv. excess probes are washed with the membrane by buffer (typically by saline sodium citrate buffer) and the pattern of hybridization is visualize depending on the top of labelled that you used -- visualized through x-ray kapag gumamit ka ng radioactive material or uv light (fluorescence material) as the labeled of the DNA probes

NORTHERN BLOT

● Almost the same procedure with the southern blot

● In contrast with SOUTHERN BLOT: THE NORTHERN BLOT is the technique to detect the RNA specifically mRNA of interest
● The steps involved in the Northern analysis include:

○ RNA isolation (total or poly(A) RNA)

○ Denaturing agarose gel electrophoresis

○ Blotting, Transfer to solid support and immobilization

○ Hybridization with probe

○ Washing

○ Detection

● The transfer buffer used for the blotting usually contains formamide

● Northern blot is also known as RNA Blot, is a technique used in molecular biology research to study gene expression
○ we are not only detecting the gene in here but also we study whether the gene is expressed
○ WHEN DO WE SAY THAT THE GENE IS EXPRESSED?

■ IF the gene transcribed and or translated

■ DNA replication is NOT A PROCESS OF GENE EXPRESSION since dna replication is a process of only duplicating the DNA; that is why we

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can not say that the gene is expressed since it only made its own copy.

● Gene expression begins when the DNA is transcribed into mRNA, and that mRNA that carries the genetic code is translated to

PROTEIN.

○ How to know there is a gene expression:

■ performed a method that detects the presence of RNA, and that is

NORTHERN BLOT

● Northern blotting involves also the use of gel electrophoresis to separate the RNA sample by size
● It is also used hybridization probe (either DNA or RNA probes; as long as complementary yung probes to the target RNA)
● The termed northern blot refers to specifically to capillary transfer of RNA from electrophoresis gel to the blotting membrane (nitrocellulose or nylon)

● The northern blot technique was developed in 1977 by James Alwin, David Kemp, and George Stark at Stanford University
● The probe that is used in here is either RNA or DNA

○ RNA that transcribed into vitro or able to withstand more rigorous washing steps preventing some of the background noise
○ Commonly cDNA is also used in northern blotting, and their probes are labeled with radioisotopes or fluorescent materials

STEPS on Northern Blot

○ ISOLATING THE RNA -A general blotting procedures begin with the extraction of total RNA from homogenized tissue sample or from the cells

■ Out of the total RNA pwede reng ispecify kung mRNA lang ba or kasama yung ibang type ng RNA
■ If in the procedure is we only want analyze mRNA

● Then we can isolate the eukaryotic messenger RNA using the oligodinucleotide containing thymine - will bind to poly-A tail of the mRNA

● RECALL: The tail part of the process mRNA in the eukaryotic cell contains lot of adenine residue (2-20 base pairs)

● In order to isolate the mRNA containing poly-A tail

○ We will use oligodinucleotide containing its complementary base which is thymine

■ The mRNA are now separated by gel electrophoresis

● To sort out the RNA according their size

DENATURATION STEPS

■ Just like in DNA double strand pinag hihiwalay naman yung RNA double regions OR double stranded regions
■ Although RNA are single stranded; may part ng RNA that forms double stranded regions - due to complementary base pairings
■ see to it that is linear when step up in denaturation

● instead of adding alkaline solution or sodium hydroxide -- it uses formaldehyde to denature the double stranded region of mRNA transcript

followed by BLOTTING TECHNIQUES

■ RNA samples now are separated by size and transfer to the nylon membrane through capillary action of the buffer with nylon membrane with the positive charge most effective for use in northern blotting (hindi na nitrocellulose ang gamit dito) nylon ang gamit dito

■ since the negatively charge of NA acid have a high affinity for RNA

■ The transfer buffer use for the blotting is usually contains FORMAMIDE - because it lowers annealing of temperature of the probes RNA interaction thus, eliminating the need for a high temperature - which can cause RNA degradation
● WHAT WAS RNA TRANSFERRED TO THE MEMBRANE?
○ It is immobilized through covalently cages to the membrane by UV LIGHT or HEAT - there is also baking steps here which is typical of the southern blot steps

■ The membrane containing RNA is incubated in the oven with 70-80°C temperature for several hours
■ fixed and immobilized na si RNA in the membrane

HYBRIDIZATION

■ After the probes has been labeled , the probe is mixed with RNA in the membrane
■ then the probe will hybridize the RNA on the membrane

WASHING STEPS

■ Tatanggalin yung unbound probes

■ the membrane is wash to ensure the probes has found its specifically and to prevent background signals

DETECTION

■ Visualize to detect whether there is a hybrid form; the hybrids signals will be detected by X-RAY FILM (if you are using radioisotopes in the probe or uv light if using fluorescent material)
● This is the way we separate the mRNA in the sample
● One of the convenient techniques for extraction of mRNA is the use oligodeoxythymine (oligo dT) it contains of thymine couple to a solid support
● oligodeoxythime hybridizes the poly-Adenilated tail which is presence on most eukaryotic mRNAs
○ that will allow the mRNA to be collected
○ and separate the impurities(proteins, partial transcript, double stranded RNAs - not need in northern blotting) other types of mRNAs

APPLICATIONS OF NORTHERN BLOT

● Northern blotting is used as a sensitive test for the detection of transcription of DNA fragments that are to be used as a probe in Southern blotting.
● It also allows the detection and quantification of specific mRNAs from different tissues and different living organisms

○ we will be able to know how much of that gene is expressed
■ ex. cancer - cell has mutation in a gene then that gene is expressed that is why there is cancer; then

you can determine the progression of the disease or its prognosis by quantifying the number of specific mRNA of that kind cancer
● so pag marami kang nameasure na mRNA you can conclude that there is too much expression of that gene - that is the sign that the cancer is severe

Northern blotting

○ is used to detect the gene expression

■ Although we have that gene, is that gene expressed?

■ we can have oncogene in your body, is that oncogene active? Will it cause cancer? will it stay there or remain dormant for the rest of your life?

● we have to know

● oncogene - is a cancer gene, if active: most likely there is transcription happening, there is gene expression
■ only northern blot allow us to identify if the gene is actively expressed; if the DNA is transcribed into mRNA

WESTERN BLOT

● Almost the same with southern blot

● Western blot - detects the proteins, a mixture of a proteins are separated by electrophoresis and transferred to nitrocellulose membrane
○ The membrane is incubated with antibody as a probe

● The probe in WESTERN BLOT is antibodies

○ The antibodies are directed against the target proteins and followed by another antibody (the antibody that will allow us to detect the presence of target proteins)

the steps are (Western Blot)

○ The electrophoresis

■ to sort out the protein size then from the gel is transferred to the nitrocellulose membrane (same with southern blot)

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■ After the proteins has been transferred to the membrane

● then add the primary antibody (first antibody), that will bind to the target proteins
○ WASHING STEP

■ To wash off the excess unbound antibody

○ ADD THE SECONDARY ANTIBODY

■ Has affinity to the primary antibodies, aside having affinity to the primary antibody; it is carrying label (either radioisotopes or fluorescence material)

■ If there is a target proteins in the nitrocellulose membrane, the primary antibody for that protein will bind with secondary antibodies

● This secondary antibody has a affinity to the primary antibody

■ The secondary antibody will be labeled with a radioisotopes or fluorescent material
○ DETECTION

■ Visualize the target proteins in labeled with these antibodies - we can performed x-ray if radio labeled the secondary antibody
● or visualize the results by uv light if you are using fluorescent labeled secondary antibodies
● The target proteins levels can be also estimated depending on the thickness of the band that forms
○ thick band - several proteins

○ thin band - less protein

● Western blot is the confirmatory test for HIV TEST

● Southern blot

○ probes: DNA probes

● Northern blot

○ probes: RNA or DNA probes

● Western blot

○ probes: primary and secondary antibodies

SIMILARITY: (Blotting Techniques)

○ ALL these probes are labeled with either radioactive materials or fluorescent material
○ depending on the labelled (kung anong detection system ang ipeperformed mo)
■ X-ray if the probes are labeled with radioisotopes

■ UV LIGHT if fluorescent materials

○ ALL of them require the gel electrophoresis, blotting steps, and hybridization steps
○ All of them are very simple and usually consist of 4 separate steps such as SIMPLIFY PROCEDURES: electrophoretic separation, protein, NA acid fragments, transferred to, immobilization on the membrane (nylon or nitrocellulose), binding of the analytical probes with the target molecules, hybridization steps, and finally the visualization of the bound probes

TECHNIQUES DERIVED FROM SOUTHERN BLOT

● Introduced by MARY LOU PARDUE and JOSEPH GALL at Yale University in 1969.

○ it has developed into a variety of genetic technique that are applied to the chromosomes preparations
○ to know the specific location of the gene in the chromosomes we can performed Fluorescence In-situ hybridization technique
○ this is applied to chromosomes preparation in metaphase or interphase nuclei
○ convention of metaphase chromosomal analysis can detects the laws or regained the materials through KARYOTYPING
■ but it should be obvious yung abnormalities

■ karyotyping - can detect the gained or loss of chromosomal materials of about 4 million base pairs or more
■ ex. we loss 1 million of base pairs, then it is not apparent to karyotyping; the abnormalities cannot be seen easily since 4 mil. up lang yung nakikita sa karyotyping

● then we will be needing more sensitive and specific method in order to detect the chromosomes abnormalities specifically to the small abnormalities (small deletion, rearrangement,translocation)
■ PRINCIPLES: rest om using the metaphase or in interphase cells and hybridizing the labeled DNA to single stranded chromosomal DNA at one or several defined sites directly to the microscope slides.

● In this technique, fluorescently labeled DNA probes are used to screen the interphase or metaphase spread of chromosomes on a glass slide
● The technique is useful in determining the specific chromosomal localization of genes and other markers

PRINCIPLE OF FISH

■ DNA probe + fluorophore

● FISH REQUIRED: labeling the DNA probe with fluorophore to make the signal visible
● in metaphase or interphase, maintaining the (metaphase or interphase )chromosomes fixed on the slides are denature into single stranded DNA - it should be single stranded to bind the complementary pairs of DNA probe

● (in the picture) the color orange biotin in the pic

○ Biotin - serves as the binding sites for fluorophore materials

● This DNA Probes(yung right na may biotin) hybridize in situ in its specific sites in the chromosomes
○ This sites is visualize by fluorescent by dark field microscopy (yung 3 row sa left)
○ by binding the fluorescence light to the labelled antibody-for biotin this antibody is a streptavidin to the biotin (the primary antibody) - to enhance the intensity of the fluorescence the secondary antibody (4th row) - it is biotinylated anti avidin antibody- it will attach with the primary antibody

○ the resulting signals will be amplified by attaching additional labeled antibodies (look on the 6#)

■ Fluorescence in situ hybridization (FISH) is a laboratory technique for detecting and locating a specific DNA sequence on a chromosome
■ The technique relies on exposing chromosomes to a small DNA sequence called a probe that has a fluorescent molecule attached to it. The probe sequence binds to its corresponding sequence on the chromosome.

EXAMPLES OF FISH IN METAPHASE

● Chromosomes are stained dark blue by DAPI stands for - (4',6-diamindino-2-phenylindole)
○ stained whole and all of the chromosomes color blue

○ EXCEPT: 5 chromosomes that carrying a fluorescent signal

■ two copies of chromosomes 3 are identified by 2 red signals

■ green fluorescent probes that hybridize the end of the short arm of chromosome 3 further green signal is visible over the long arm of chromosome 16

● and these green signals - indicates the presence of 3 chromosomal segments of the long arm of chromosome #3 there could be a trisomy if there is 3 chromosomal segments

● DNA probes are specific to chromosomes number they are specific to chromosome segments- then you will know what chromosome is involved or what chromosome number fluoresce

● On the right side pic is the interphase FISH, two green signals identify chromosome #22- the probe here is specific for chromosome 22

○ red signals - signals the long arm

■ the upper cell (on the pic) indicates normal

■ the lower cell (on the pic) shows a lack of red signals - that indicates the loss or deletion of long arm chromosome 22
● That is how we use the FISH method

EXAMPLES OF FISH IN METAPHASE

● FISH of telomerase sequences

● Telomeric sequence: TTAGGG

○ the telomeric sequence is repeatedly by 3,000X

○ which can reach 15,OOO bp in length

● In this human metaphase, all telomeres are labelled with probes that hybridizes specifically to the telomeric sequence of the chromosomes
● two signals are visible in each telomere (one each chromatin) end to end

● kaya visible sya once labeled with DNA probes - maraming base sequences with

TTAGGG

○ Most likely the ano yung sequence ng DNA probes nya?

■ Dapat complementary sequences with the telomeric sequence it may contains AAUCCC the sequence of dna probes
■ And that dna probes are labeled with fluorescent kaya nakikita yung fluorescent which indicates the location of the telomere

DOT BLOT

● Dot blot - or slot blot, is a technique in molecular biology used to detect proteins
● REPRESENT THE: Simplified western blot version

○ With an exception that the proteins are detected or not

○ No more electrophoresis - not required

○ The samples are applied directly on the membrane in a single spot (either of nitrocellulose or nylon membrane)
○ The blotting procedure are included

● Derived from: western blot because they have similarities as that of western blot
● It is a western blot BUT WITHOUT ELECTROPHORESIS

● The technique offers a significant savings of time since no more gel electrophoresis
● The complex blotting for the gel are not required however , it offers no information about the formation of size of the target proteins
● In this picture is the general step for dot blot technique

● Dispensing of microliter samples directly into membrane (nitrocellulose)

● Let it dry couple of minutes
● The samples can

be in the form of tissue culture, supernatant, blood serum, cell extract - kumbaga crude samples directly from the patients

● No need to purify

● The membrane is incubated 70-80°C to immobilize the molecule (to fixed/attach the proteins in the

membrane BAKING THE MEMBRANE)

● It is incubated with primary antibody followed by the detection or secondary antibody similarly with western blot