Blotting techniques Flashcards
formation of hydrogen bonds between to complementary strands of nucleic acid
hybridization
during the annealing process, both — are not labeled with any isotopes or fluorescence. however, if one strand is labeled, this labeled strand is referred to as — and the process is called —
both nucleic acid strands are not labeled
labeled strand = probe
process = hybridization
hybridization reaction that is used to analyze the nucleic acid content of an unknown sample
hybridization assay
enumerate the hybridization assays
- southern hybridization
- northern hybridization
- dot/blot hybridization
- microarray
- fluorescent in situ hybridization
this hybridization facilitate gene mapping through restriction of mapping of genes and detection of restriction fragment length polymorphisms
SOUTHERN H.
(DNA)
this is used to identify homologous sequences in genomic DNA
SOUTHERN H.
arrange the process of southern h.
- if those fragments are visualized by staining, the gel should show a series of smear bands without any discrete, distinguishable bands.
- followed by one or a few specific restriction enzyme digestion. enzyme digestion will produce thousands of fragment of genomic dna.
- purify genomic dna from eukaryotic cells or bacteria.
- dna fragments are separated by agarose gel electrophoresis which separates the fragments according to size, the small dna fragments migrating farthest in the electric field.
- after gel electrophoresis, dna fragments are further fragmented to smaller than 1 kb size and denatured by mild alkali solution followed by transfer.
- a labeled probe is added to hybridize with the fragments on the membrane for detection and identification.
- purify genomic dna from eukaryotic cells or bacteria.
- if those fragments are visualized by staining, the gel should show a series of smear bands without any discrete, distinguishable bands.
- dna fragments are separated by agarose gel electrophoresis which separates the fragments according to size, the small dna fragments migrating farthest in the electric field.
- if those fragments are visualized by staining, the gel should show a series of smear bands without any discrete, distinguishable bands.
- after gel electrophoresis, dna fragments are further fragmented to smaller than 1 kb size and denatured by mild alkali solution followed by transfer.
- a labeled probe is added to hybridize with the fragments on the membrane for detection and identification.
summary of the process of southern h.
in order
- extraction & purification
- enzyme digestion
- DNA denaturation & fragmentation
- transfer / blotting
- hybridization with labeled DNA probe
- detection & identification
enzyme digestion product = DNA fragments
parameters of southern h.
- resolution of the agarose gel
- depurination of DNA molecules
- physical transfer of DNA onto the membrane support
- fix DNA onto the nylon membrane
southern h.
these paramaters depent on the resolution of the agarose gel which include the — of the agarose gel used and the — applied in gel electrophoresis
percentage and voltage
resolution of the agarose gel - southern h.
percentage of the agarose gel used and the voltage applied in gel electrophoresis
percentage: 0.7 - 1.2%
high voltage combined with short run times: 10 - 20 kbp
low voltage combined with long run times: 1-10 kbp
paramaters of southern h.
the most critical step which these shorter fragments are more efficiently transferred in the blotting
depurination of DNA molecules
depurination by — upon denaturation by sodium hydroxide leads to nicks in the DNA strands, resulting in a breakdown of long DNA fragments into shorter pieces of single-stranded DNA
0.2 M hydrochloric acid
physical transefer of DNA onto the membrane support
enumerate the 2 diff. transfer methods
- ascending capillary transfer (classical southern transfer)
- descending capillary transfer
ascending capillary transfer
- nylon membrane is place on top of agarose gel
- agarose gel is placed on top of a solid supporter in the presence of salt (identify what salt)
arrange in order
- agarose gel is placed on top of a solid supporter in the presence of salt 10 saline-sodium citrate (SSC buffer)
- nylon membrane is place on top of agarose gel
this arrangement allows efficient diffusion of DNA onto the nylon membrane
this uses the gravitational flow of the transfer buffer with the help of vacuum to allow more rapid and reproducible blotting of DNA
Descending capillary transfer
why nylone membrane is used in descending capillary transfer?
bcos it has high DNA/RNA binding capacity and strong mechanical strength on stropping and reprobing
these methods are effective for fixation of DNA to a nylon membrane
- 80C for 2 hours
- UV light irradiation
true or false
the baked or UV-fixed southern blot cannot be stored for prolonged periods of time at RT
FALSE.
it can be stored
this allows detection of a given RNA molecules in a mixture of heterogenous RNA
NORTHERN H.
(RNA)
why denaturation is important in northern h.?
because native RNA tends to form secondary structure
so it has to be denatured b4 and during electrophoresis
northern h.
before electrophoresis, denaturation is achieved by?
heating the sample to 55C in the presence of formaldehyde and formamide
northern h.
during electrophoresis of RNA, addition of formaldehyde to the gel prevents —
reformation of secondary structure
northern h.
decrease the hybridization signal compared with unstained RNA as it can decrease the hybridization signal compared with unstained RNA
ethidium bromide
northern h.
after electrophoresis, the separated RNA can be transferred onto a nylon membrane through —
capillary blotting or vacuum blotting
northern h.
RNA can be fixed by —
UV light irradiation or baking at 80C
enumerate the step-by-step process in northern blotting
- denaturation
- addition of formaldehyde during electrophoresis
- transfer the separated RNA in a nylon membrane after electrophoresis
- Bake or UV fix the RNA
this results from a variable number of tandem repeats in a short DNA segment
Restriction Fragment Length Polymorphism (RFLP)
enumerate the uses of RFLP
- DNA fingerprinting
- paternity testing
- genetic disease marker
this is performed when genomic DNA is collected and is digested with a specific restriction enzyme followed by gel electrophoresis.
RFLP analysis
— can be used to amplify very small amounts of DNA in 2-3 hrs to the levels required for RFLP analysis. therefore, more samples can be analyzed in a shorter time. this approach is known as —
PCR
approach: Cleaved Amplified Polymorphic Sequence Assay (CAPS)
this permits the simultaneous evaluation of hundreds to thousands of different DNA regions distributed randomly throughout the genome without prior sequence knowledge
Amplified Fragment Length Polymorphisms (AFLP)
useful in nonmodel species which have no complete genom sequences available and where other types of genome-wide markers such as SNPs and microsatellites are difficult to obtain
AFLP
enumerate the step-by-step AFLP analysis
- generation of restriction fragments by using two different restriction endonucleases
- preselective amplification
- selective amplification
- visualization (PAGE/CE)
1st step of AFLP analysis
enumerate the 2 diff. restriction endonucleases
the digested DNA fragments are ligated to — that contain a known core sequence of approx. 20 nucleotides
- rare cutting frequency
- high cutting frequency
double stranded oligonucleotide adapters
identify what step of AFLP analysis
in this step, the sequences of the ligated adapters serve as primer binding sites for PCR of all the fragments derived from cutting with both enzymes which are called —
PRESELECTIVE AMPLIFICATION
heterosite restriction fragments
identify what step of AFLP analysis
this step provides a higher amount of template DNA for subsequent rounds of more selective AFLP reactions
preselective amplification
AFLP analysis
2 methods of visualization
- conventional denaturing polyacrylamide gel electrophoresis (PAGE)
- capillary electrophoresis (CE)
this combines the specificity of RFLP with the sensitivity of the PCR which allow high resolution genotyping of fingerprinting quality
AFLP analysis
in terms of time and cost efficiency, replicability and resolution of AFLPs, which is better RFLP or AFLP?
AFLP
enumerate the 5
AFLP markers is a major type of genetic marker used in —
- systematics
- pathotyping
- population genetics
- DNA fingerprinting
- quantitative trait locus mapping
this must be labeled with a radioactive or other type of marker. this is denatured by heating and applied to the membrane in a solution of chemicals that promote nucleic acid hybridization
probes
short, single or low copy genomic DNA or cDNA clones
RFLP probes
enumerate the 4
RFLP probes are used in genome mapping an in variation analysis including —
- genotyping
- forensics
- paternity tests
- hereditary disease diagnostics
this refers to a labeled DNA sequence that hybridizes with one or more fragments of the digested DNA sample after they have been separated by gel electrophoresis
RFLP probes
this carry a radioactive isotope of phosphorus, 32 p
labeling with a radioactive marker
enumerate the methods in labeling with a radioactive marker
for detection: —-
METHODS:
- Nick translation
- End filling
- Random priming
detection: autoradiography
the specificity of the hybridization signal in southern and northern blotting is mainly determined by the —
hybridization temperature
the higher the hybridization temperature, the more stringent.
in this method, a sheet of X-ray sensitive photoraphic film is placed over the membrane…
autoradiography
deoxyuridine triphosphate (dUTP) nucleotides by reaction with biotin, this refers to —
labeling with a nonradioactive marker
biotin is an organic molecule that has a high affinity for a protein called —
avidin
labeling with a nonradioactive marker
the probe DNA is complexed with the enzyme — and is detected through the enzyme’s ability to degrade — with the emission of —
the probe DNA is complexed with the enzyme horseradish peroxidase and is detected through the enzyme’s ability to degrade luminol with the emission of chemiluminescence
detecting nucleic acids with a nonradioactive probe, indirect or direct?
indirect
the target is spotted in duplicate, side by side. the last two rows of spots contain positive and negative control, followed by a black with no target. this refers to —
dot blot
the top rows of the — gel on the left represent positive and negative control followed by four samples spotted in duplicate blank with no target are in the last four spots on the right. this refers to —
slot blot
the two bottom rows of slot blot represent a — that is often useful to confirm that equal amounts of DNA or RNA were spotted for each test sample
loading or normalization control
a variation of the dot/slot blot in which the dotted material is arranged in a regular gridlike pattern with each feature reduced to a very small size so that hundres to thousands of probes can be placed on one solid surface
microarray
most common application of microarray technology
transcript profiling
process of transferring/blotting the proteins from the gel onto the surface of an absorbent membrane to render the proteins accessible for the identifying ligand
WESTERN BLOT
(protein)
enumerate the 2 methods of gel electrophoresis for protein separation
- SDS-PAGE
- Isoelectric focusing (IEF)
Gel electrophoresis for protein separation
in this method, proteins are fully denatured with SDS and heat in the presence of B-mercaptoethanol
SDS-PAGE
Gel electrophoresis for protein separation
in this method, native proteins are separated in a gel containing a pH gradient and works well for hydrophilic proteins.
Isoelectric focusing
Gel electrophoresis for protein separation
hydrophobic or amphiphilic proteins require detergents to stay in solution durin separation, this refers to —
Isoelectric focusing
enumerate the methods for transferring protein onto membranes
- Diffusion (passive transfer)
- adided by capillary action or vacuum
- electrotransfer
transferring protein onto membranes
this can be achieved by simply posing a membrane sheet on one or both of the gel surfaces.
passive transfers
(DIFFUSION)
transferring protein onto membranes
this method is achieved by putting the gel-membrane sandwich in a suitable holder and immersing it in a tank filled with buffer and fitter with two plate electrodes
electrotransfer
transferring protein onto membranes
this requires no buffer tank and no cooling system and several gels can be processed simultaneously and the transfer time is shorter than with tank blotting
semidry blotting
transferring protein onto membranes
electroblotting is fast and gives a sharp replica of the gel. the strong electric force can cause —
BLOW THROUGH
some proteins may not be retained by the membrane
enumerate the widely used membranes used for protin blotting
- nitrocellulose
- nylon
- polyvinylidene difluoride (PVDF)
most commonly used membrane for protein blotting, it has satisfactory capacity, and gives low bg but is fragile.
nitrocellulose
this membrane has much superior mechanical strength and its protein binding capacity is good.
it can be obtained as positively charged membrane that even increases the binding and retention capacity.
but not suitable for total protein stain
nylon
membrane of choice for many applications of western blotting
PVDF
this membrane combines high protein binding capacity with excellent mechanical resistance and good staining properties
PVDF
standard stains of similar sensitivity
amino black & india ink
the stain with lowest sensitivity and it is convenient for many applications as it is reversible by briefly immerding the stained blot in —
PONCEAU
0.1 N NaOH
protein bands on this membrane become translucent on immersion in methanol and thus can be visualized on a lightbox
PVDF
enumerate the stain/s used for both NC and PVDF
- Amido black
- Colloidal gold
- India ink
- Ponceau red
this stain/s is used only for PVDF
Coomassie & Transillumination
enumerate the blocking agents
- ovalbumin/gelatin
- fat free milk
- tween-20
blocking agent
this gives optimal blocking with lowest bg and without impairing immunoreactivity of the blotted proteins.
time and temp: 60 min at 40C
ovalbumin/gelatin
blocking agent
this proved to be an economical and effective blocker in most systems.
time and temp: 30 min at RT
fat free milk powder
blocking agent
very simple and has a tendency to give elevated background. it may mask or detach immunoreactive proteins in some occasions.
time and temp: 30 min at 37C
tween-20
immunodetection of specific proteins in western blot is usually done by the — method. this involves — (enumerate)
indirect method
poly- or monoclonal antibody directed against the protein on the blot (primary antibody) and a labeled antispecies antibody (secondary Ab) or labeled protein A or G
most commonly used enzymes for secondary antibody
- horseradish peroxidase
- alkaline phosphatase
this system provides a higher sensitivity than the color production system
chemiluminescence (CL) system
factos that influence hybrid stability
- ionic strength
- base compositon
- destabilizing agents
- mismatched base pairs
- duplex length
enumerate the influence
- ionic strength: melting temp increases for each 10 fold increase in monocovalent ions
- base compositon: GC base pairs are more stable than AT base pairs
- destabilizing agents: every 1% of formamide decreases the melting temp by around 0.6C for DNA hybrid. 6M urea decreases the melting temp around 30C
- mismatched base pairs: melting temp decreases by 1C
- duplex length: no detectable effect with probes greater than 500 bp
factors that influence hybdridization rate
emumerate the factors
- temp
- ionic strength
- destabilizing agent
- mismatched base pairs
- duplex length
- viscosity
- probe complexity
- base composition
- pH
kamo na dayn bahala sa influence ~
