Techniques of Mol bio

Dr. Anwar Hussain

Professor
Department of Botany
DNA
Gel Electrophoresis
• Gel is--- agarose (50 to 20kb)
• or polyacrylamide (5-500bp)
• Mechanism of separation not well understood
• Migration of fragment through pores of matrix—important
• In free solution migration is independent of MW
• Agarose—complex network of polymeric substances
• Pore size depends on buffer conc, type and conc of agarose
• DNA molecules during GE contract into balls
• Smaller balls pass through pores, Larger by reptation
DNA size Marker

100 bp 1 Kb
Gel loading dye
• 25 mg of bromophenol blue to 6.7 ml of ddH2O and mix.
• 25 mg of xylene cyanol FF and mix.
• 3.3 ml of glycerol and mix.
• Aliquot and freeze at -20 °C for long-term storage.
• Ethidium bromide
• CYBR Gold
• CYBR Green
• CYBR safe
• Eva Green
Gel Electrophoresis
Bigger DNA molecules (greater globular volume than pore size) can only pass through by reputation
• Occurs with molecules about 20 kb
• Difficult to separate molecules larger than this with common GE
• Pulsed electrical fields in pulse field gel electrophoresis
• Upto 10 mb can be separated
• Regular changes the orientation of electric field
• Cause DNA to re-align before migration
• Electric field parameters (duration, intensity and direction) set each time
• Major disadvantage DNA path not straight
• Contour-clamped homogenous electrical field gel electrophoresis (CHEF)
• Previous—re-orientation angle 120
• Recent angle can vary 106 for whole yeast chromosomen
Gel Electrophoresis
Ethidium bromide/SYBR SafeTM
bromphenol blue in a 50% glycerol solution
Ladder
0.05 µl DNA in one band
Illuminated in UV (transilluminator)
GE used for Protein-NA interaction (gel retardation or band shift assay
Applications
➢Resolving DNA fragments of different
lengths
➢separate different molecular configurations of
a DNA molecule
➢investigating protein–nucleic acid
interactions in the so-called gel retardation or
band shift assay
Polyaccrylamide Gel Electrophoresis
• PAGE for proteins and smaller fragments of DNA
• Movement is determined by the charge, size (molecular weight) and shape
of the molecule
• Unlike DNA and RNA, proteins vary in charge according to the amino acids
incorporated, which can influence how they run
• Amino acid strings may also form secondary structures that impact their
apparent size and consequently how they are able to move through the
pores
• It may therefore sometimes be desirable to denature proteins prior to
electrophoresis to linearize them if a more accurate estimate of size is
required.
•
SDS PAGE vs native PAGE

• Denaturing or non-denaturing conditions, depending on the purpose of the

analysis.
• Sodium dodecyl sulphate (SDS), in combination with heat and sometimes a
reducing agent is used to denature proteins
• The heat disrupts the hydrogen bonds that hold secondary and tertiary
structures
• Reducing agent, such as β-mercaptoethanol, cleaves disulfide bridges
• Proteins are linearized and complex with the SDS so that all have a similar
mass-to-charge ratio
• This eliminates the influence of structure and charge, and proteins are
separated solely on the basis of differences in their molecular weight
• Typically used to separate proteins of 5–250 kDa.
Native PAGE
• In native PAGE preserves the protein’s higher order structure
• Consequently, the distribution of proteins through the gel is mainly
influenced by the protein’s charge and the pH of the separation
• It allows researchers to analyze proteins in their natural or “native”
state.
• Desirable when analyzing bound proteins or complexes
• Lower voltages may be used for separation.
•
Principle of PAGE
• Ammonium persulfate (APS)
and
tetramethylethylenediamine
(TEMED) are used to catalyze
polymerization
• A net like structure is formed
• Coomassie brilliant blue is
used for protein staining on
gel
Stacking and Resolving Gel
• The role of the stacking gel is to allow sample loading and make sure that all the
samples enter resolving gel at the same time
• The proteins within the sample will then be separated so they can be “resolved”
in the resolving gel.
• The optimal gel percentage will depend upon the sizes of the proteins to be
separated
• The lower the percentage, the larger the proteins that can pass through
• A lower percentage gel (often around 4% total acrylamide) is used for the
stacking gel irrespective of analyte size as it does not perform the separation.
• It is normally at a lower pH (around 6.8 compared to 8.8) than the resolving gel
• The percentage of the resolving gel is varied depending on the size of the
proteins you wish to separate, typically falling between 7 and 12%.
Protein footprint assay Band shift assay
Lac repressor bind to
DNA protect it against
the Dnase I leaving a foot
print (missing fragments)
Restriction mapping
Test your knowledge
 Test your knowledge
A B Mix

9 Kb A 9 Kb
9 Kb

6 Kb B B

3 Kb 6 Kb 6 Kb 6 Kb
 Test your knowledge A

A B C Mix

20 Kb

B
15 Kb

10 Kb

7.5 Kb

5 Kb
B
2.5 Kb
Nucleic acid blotting
➢Immobilization of DNA
➢Blotted NA used as target
Types:
➢Blotting of NA from gel
➢Dot ant spot
➢Colony and plaque
Nucleic Acid Hybridization
• Hybridization may be applied in the isolation
and quantification of specific nucleic acid
sequences
• In the study of Nucleic acid organization,
intracellular localization, expression, and
regulation.
• Other applications includes the diagnosis of
infectious and inherited disease.
Capillary Blotting apparatus

Pre-treatment: On gel short depurination

treatment (0.25 mol/l HCl)
followed by alkali

Ensures:
1. Transfer of a wide range of DNA by
shortening long fragments
2. Denaturation
Southern Blotting
Stringency control in
Hybridization
• The specificity with which a particular target sequence is detected by
hybridization to a probe
• Most common control is by the temperature and salt concentration in
the post-hybridization washes
• Stringency washes are performed under successively more stringent
conditions (lower salt or higher temperature)

• M = ionic strength of buffer in moles/liter

Stringency Washes
• The “Wallace rule” (Lay Thein & Wallace 1986) is used to determine
the appropriate stringency wash temperature:
• For oligonucleotide probes, the hybridization step is usually
performed at 5°C below Tm
• For every mismatch, a further 5°C reduction is necessary to maintain
hybrid stability.
Colony hybridization
Plaque
hybridization
DNA Amplifiction
Primer
Polymerase chain reaction
Long Accurate PRC
➢ No 3 ′ –5 ′ exonuclease (proofreading)
activity in Taq polymerase.
➢ To overcome this
5′-ATCGCGGCTTAAGCAATGTC----------AGTCAACGATTACTTGCAT-3′

A) 5′-TAGCGCCGAACGTTACAG-3′
B) 5′-TCAGTTGCTAATGAACGCTA-3′
Emulsion PCR
• PCR components, such as template DNA, polymerase, primers,
nucleotides and minerals are soluble in water but not oil
• This forms the basis for the concept behind emulsion PCR, which is a
technique that amplifies DNA molecules in physically separated
water-in-oil droplets
• Both Ion Torrent and GenapSys sequencing platforms utilize the
approach.
• The DNA is fragmented into small pieces and purified
• Adaptors are ligated at the ends of these fragments
• Beads are added to the mixture containing immobilized primers
• The aqueous solution (now containing the library of DNA fragments, a
large number of prepared beads and the necessary PCR reagents) is
emulsified in oil from very small droplets
• The droplets contain a bead and a single DNA fragment. The DNA is
replicated by DNA polymerase after one of the ends of the fragment
anneals to its complementary oligonucleotides covalently attached to
the bead.
Bridge Amplification
• DNA is fragmented and adapters are linked to it
• The fragments are immobilized to a flow cell which is a class slide
composed of lanes
• Each lane is a channel composed of a lawn of two types of oligos
• The fragment strand hybridize to the oligos on the flow cell
• Polymerase amplify the attached fragments and the original strand is
washed away
• Then the strand folds over and the adapter region hybridizes to the second
type of oligo on the flow cell
• Another round or amplification amplify the fragment resulting in a double
stranded bridge
• The double stranded bridge is denatured
• The process is then repeated
Whole Genome Sequencing
• Five different methods of WGA have been developed
• Four of them are direct variants of PCR generate Short length
products (<1000 nucleotides)
• they have not been widely adopted
• Multiple displacement amplification (MDA) Does not involve PCR
• The template is replicated again and again by a hyperbranching
mechanism of strand displacement synthesis
• the polymerase lays down new copies of the template concurrently
with the displacement of new copies
• Uses DNA polymerase from bacteriophage ϕ 29
Steps MDA