VALIDATION
ANALYSES PART 1
(Restriction Analysis -Practical 3)
Introduction
What have you done so far?
Ø At the end of MOLECULAR CLONING, you obtained:
1) Purified plasmids from different bacterial colonies à plasmid clones
ü 3 types of plasmids, each from different colony on 1:2 transformant plate. 1 positive
control plasmid (Clone 4)
2) Validated for purity and quantity of these plasmid via Nanodrop
Ø But before you can use any of these plasmids, you need to validate whether it can be used
for the second phase of recombinant protein expression:
1) Preliminary Analysis – quick determination of presence of insert in all plasmid clones
ü Restriction Endonuclease Digestion Analysis (Restriction Analysis) – identify positive clone(s)
2) Confirmatory Analysis – to finally establish with certainty whether plasmid clones are
suitable
ü Sequencing Analysis – one of the positive clones identified in the preliminary analysis
Introduction
What is Restriction Analysis?
Restriction Analysis is an analytical procedure that involves two processes:
Ø Perform restriction digestion of plasmid sample clones with correct Restriction
Enzymes
ü able to diagnose the presence of insert
Ø Perform agarose gel electrophoresis of the digested plasmid clones
Ø Important to have appropriate control(s) for diagnostic comparison
Restriction Analysis – Restriction Digestion
Nucleases
Ø There are different types of nucleases as shown here
Ø Endonucleases can:
a) either cleave randomly with the DNA or
b) can cleave at specific DNA sequences à Restriction Endonuclease
Ø Of interest to Restriction Analysis is the Restriction Endonucleases
Restriction Analysis – Restriction Digestion
Restriction Endonucleases (R.E.s)
Ø Enzymes that catalyzes the cleavage of DNA at specific sites within the DNA
Ø R.E.s: Nomenclature & Types:
*Refer to Supplementary Notes
Restriction Analysis – Restriction Digestion
Restriction Endonucleases (R.E.s)
Ø
Of interest to Restriction Analysis is the Restriction Endonucleases Type 2
q recognize short specific sequences on duplex DNA (4, 5, 6, 8
or 12 bp)
q cleave DNA strands within or close to the recognition site
q cut a DNA molecule by cleaving the phosphodiester bonds
between two internal residues
q recognize target site as a dimer
q recognition sequences are the same on both strands
à palindromic sequence
q generate termini bearing 5’ – phosphate and 3’ – OH
• blunt ends – no nucleotide overhangs
• sticky ends – also called protruding, staggered, cohesive ends
à 5’ overhang (e.g. EcoRI and HindIII)
à 3’ overhang (e.g. Pst1)
2 subunits
of R.E.
Restriction Analysis – Restriction Digestion
Restriction Digest
What are the four possible outcomes of a restriction digest?
(1) Complete digestion
(2) Partial digestion
(3) Star Activity
(4) No digestion
(1) Complete digestion
Ø
The cleavage products should be present in EQUIMOLAR quantities
Ø
When product stained with Ethidium Bromide and visualized
à band intensity is proportional to fragment length
à for e.g. 8-kb fragments will stain at twice intensity as 4-kb fragment
Ø
Normally, sum of the lengths of the restriction fragments should be equal to the total length of DNA
molecule
à need to check for co-migration of fragments of equal length (identified by increased band intensity)
Restriction Analysis – Restriction Digestion
Restriction Digest
(2) Partial/Incomplete digestion
Ø
occurs when only
q one or more restriction sites on a DNA molecule are not cleaved by R.E.(s)
q not all DNA molecules are completely cleaved (where some remains intact)
à results in more DNA fragments observed than expected
à sum of the lengths of the restriction fragments LARGER than the total length of DNA molecule
ü Complete digestion of this DNA molecule
should produce only 4 digested fragments
corresponding to fragments that are *
*
ü Incomplete or partial digestion would result
in additional digested fragments
( those fragments that are not * )
*
ü the number indicated in < > the fragment length in kb.
Total length of DNA molecule is 5 + 9 + 5 + 3 = 22 kb
*
*
ü But in incomplete digestion, total sum of restricted
fragments will > 22 kb
Restriction Analysis – Restriction Digestion
Restriction Digest
(2) Partial/Incomplete digestion
Ø Reason for its occurrence?
a) sub-optimal condition
b) methylation
Ø Sub-optimal condition can result from:
•
•
•
•
•
•
bad quality or insufficient amount of R.E.s
inaccurate pipetting
impurities that affect R.E. activity
wrong buffer and temperature condition
incubation period too short
etc….
Ø Understand how to recognize partial digestion
q Additional low-intensity bands are present above the expected bands
Ø Partial digestion can be useful, for e.g., in generating gDNA library or restriction mapping
Restriction Analysis – Restriction Digestion
Restriction Digest
(3) Star Activity
Ø Occurs under non-standard conditions where the R.E. relax their recognition sequence
specificity and cleave at similar recognition sequence producing unexpected bands
Ø Reason for its occurrence?
a)
b)
c)
d)
e)
f)
high enzyme/DNA ratio
low ionic strength in buffer
high glycerol content in buffer
high ethanol content
high pH
Mn2+ ions used instead of Mg2+ ions
Ø Understand how to recognize star activity
q there will more and smaller bands found below the expected bands, with the intensity of the
bands not correlated to the size
Restriction Analysis – Restriction Digestion
Restriction Digest
(3) How to recognize partial digest and star activity in on a gel?
Partial digestion:
•
•
•
Additional low-intensity bands are present above the expected bands on
the gels.
No additional bands are present below the smallest expected fragment.
These additional bands disappear when the incubation time or amount of
enzyme is increased.
Star activity:
•
•
•
Complete
cleavage
Additional DNA bands on the gel are lower than the expected bands.
No additional bands higher than the largest expected fragment.
Adding more units of enzyme or increasing the incubation time only makes
the problem worse.
Restriction Analysis – Restriction Digestion
Restriction Digest
In Summary:
1)
Understand RE and type of products it can generate
2)
Outcomes of restriction digestion
3)
Factors that leads to the various outcomes of digestion
4)
How to distinguish the different types of digestion when you view the gel of digested products?
5)
How to predict the number of sites & number of digested fragments?
a) Check if template is circular or linear à are the number of digested fragments same for both or different?
b) Check if R.E. is affected by methylation
c) To predict à understand correlation between
(i) the number of bases recognised by R.E. and
(ii) length of template
à please refer to file on ”Exercise on Restriction Digestion and Methylation”
Restriction Analysis – Methylation
Methylation
Ø A number of methylases covalently join methyl groups to cytosine or adenine residues within specific
target/recognition sequences
Ø In mammalian cells, DNA methylation is essential for regulating gene expression and most commonly
occurs at a cytosine base that is immediately followed by a guanine base (annotated as a “CpG” site)
Ø Apart from regulation, methylation in bacterial cells plays an additional role. Together with restriction
modification system, it helps in protecting bacteria from foreign DNAs, e.g. viral DNAs or
transposons.
Restriction-modification systems contain a DNA methylase that protects host DNA sequences from restriction
with their cognate restriction enzymes which digest unmodified foreign DNAs. In other words, bacteria will modify
its own DNA via methylation so it is able to distinguish it from foreign DNAs à able to recognise its own DNA
Ø E. coli host strains contain the following methylases:
1) Dam (methylates adenine in the sequence G mA TC)
2) Dcm (methylates the internal cytosine residue in the sequences C mC (A/T)GG)
Restriction Analysis – Methylation
Methylation
Why methylation important in context of Molecular Cloning?
Ø AFFECT restriction digestion by:
i. completely blocking the digestion/restriction by R.E.
ii. reduce the digestion/restriction activity of R.E.
Ø
ONLY methylation-sensitive R.E.s (e.g. XbaI, StuI) will be affected while methylation-resistant R.E.s
(e.g. BglII, BstNI) are unaffected
q Not all restriction digestions are affected by methylation
q Use NEBcutter website to check for affected RE sites: http://nc2.neb.com/NEBcutter2/
Ø
How can you cut an R.E. site which is blocked by methylation?
q Prepare plasmid DNA from bacterial strain that lack methylases (e.g. Dam/Dcm deletion strains)
Ø
Consider this:
q Are following methylated?:
i. nucleotide in PCR products (e.g. in Practical 1, is overnight R.E. digestion affected?) or
ii. nucleotide in sequence filled up with DNA polymerase in vitro (Lecture 3 by Prof Lehming,
Slide 8)
Check with me for the answers!
Restriction Analysis – Methylation
Methylation
Ø How can you determine if digestion of a particular R.E. site is blocked by methylation?
q The blocking of R.E. can cognised in two ways:
i. Methylation recognition sequence can be directly seen in the R.E. site
ii. Overlapping methylation recognition sequence where you need to consider flanking sequences beyond R.E site
q Refer to ”Exercise on Restriction Digestion and Methylation” where two examples are
demonstrated with XbaI & StuI R.E.s.
q Practise with questions provided in Assignment 4
q Also, you need to understand how to calculate:
a) the probability of a particular R.E. site is methylated (and thus can’t be cut by R.E.)
b) the probable number of digested fragments obtained from a template that contains
methylation-sensitive RE sites.
Refer to ”Exercise on Restriction Digestion and Methylation” where two problem cases on Cla I
and XbaI are provided
Note that these are popular types of questions appearing in CA1.
Do attempt the Exercise and Assignment provided to you!
Restriction Analysis – Gel Electrophoresis
Gel electrophoresis
Ø Refer to “Information on Agarose Gel Electrophoresis” pdf file uploaded in Practical 1 channel in MS
TEAMS
Ø In essence, gel electrophoresis separates DNA molecules according to size where the largest - and thus
slowest moving - DNA molecules are near the top of the gel
Distance migrated by dye
Ø There is a logarithmic correlation between mobility and molecular size of DNA.
Dye front
Distance
migrated
by DNA
Restriction Analysis – Gel Electrophoresis
Gel electrophoresis
Ø They are different types of gel used for
separation of DNA molecules:
1) Polyacrylamide Gel: able to separate
small DNA molecules that differ by
ONE base (see Fig (c))
Agarose
Gel
Polyacrylamide
Gel (e.g.
Sequencing Gel)
o A polyacrylamide gel with small pores was used
to fractionate single-stranded DNA. In the
size range of 10 to 500 nucleotides, DNA
molecules that differ in size by just a single
nucleotide can be separated from each other.
In the example seen in Fig (c), the four lanes
represent sets of DNA molecules synthesized
in the course of a DNA sequencing procedure
2) Agarose Gel: used for separation of
larger DNA molecules (see Fig (b))
Ø Refer to Supplementary Notes for further
details on the figure
Ref: Molecular Biology of the Cell, 6th edition, Alberts B et al.
Restriction Analysis – Gel Electrophoresis
Gel electrophoresis – identifying Topoisomers of plasmid DNA
Ø There are three main conformations (topoisomers) of plasmid DNA:
1) Closed circular supercoiled – normally plasmid purified from E coli are negatively supercoiled
2) Nicked circular
3) Linear duplex DNA
Ø These conformations have different hydrodynamic radii where supercoiled topoisomer has the smallest
radii and nicked circular has the biggest radii
Ø These topoisomers of the same molecular weight
migrate at different rates through agarose gels:
q Supercoiling essentially winds the molecules up, giving
them a smaller hydrodynamic radius and allowing them
to pass more readily through the gel matrix
q Nicked or relaxed circular molecules that have lost all
of their superhelicity migrate appreciably slower than
either supercoils or linear molecules
q Circular DNA can form concatenated forms (dimers, trimers, etc.). Therefore, multiple bands are often
observed in highly purified plasmid preparations.
Ø Ethidium Bromide can have a pronounced effect on mobility of these topoisomers. See supplementary note
Restriction Analysis – Gel Electrophoresis
Gel electrophoresis – identifying Topoisomers of plasmid DNA
Ø In the figure below, an intact plasmid is subjected to progressive restriction endonuclease digestion
and the digests at different timepoints are run on agarose gel
à observe migratory behaviour of different plasmid topoisomers
Ø Ethidium Bromide can have a pronounced effect on mobility of these topoisomers. See supplementary note
(understand the term “critical concentration of Ethidium Bromide”)