TECHNIQUES & TOOLS FOR STUDYING DNA
Genomes are very large…
- so need methods to obtain small (relatively speaking) sections
of DNA in abundant & pure form for molecular analysis
1. Restriction enzyme cleavage & agarose gel electrophoresis
2. Southern hybridization
3. PCR (polymerase chain reaction)
4. Molecular cloning
1. Restriction enzymes - cleave DNA into specific, small fragments
1. DNA endonucleases cut
double-stranded DNA at
specific recognition sites
(often palindromic)
“blunt” ends
“sticky” ends
Fig.2.10
BamHI: staggered cut
with 5’ overhang
2. Recognition sequences often 4 or 6 bp, but also “rare cutters”
(eg. NotI 5’ GCGGCCGC 3’ ) can be useful for generating very large
fragments in genomic mapping
Sites are often shown as one strand,
but implicit that double-stranded
3. Two different restriction enzymes may generate same “sticky ends”
-“compatible ends” useful for cloning
(eg. partial Sau3A genomic digest ligated into BamHI site in vector)
4. Isoschizomers – restriction enzymes with identical recognition
sequences
… but may have different response to methylation state
MspI cleaves 5’ CCGG 3’ regardless of methylation state
HpaII does not cleave 5’ CCGG 3’ if 2d C is methylated
Example using isoschizomers to assess DNA methylation state
of genes in cancer patients
- used assay called “HpaII tiny fragment Enrichment by Ligation–mediated PCR”
- found “significant aberrancy in promoter methylation patterns
compared with normal NBCs”
NBC: naïve B cells (ie.
from healthy people)
log(HpaII/MspI) ratios
“Genome-wide DNA methylation analysis reveals novel targets for
drug development in mantle cell lymphoma” Leshchenko et al. Blood 116:1025, 2010
Agarose gel electrophoresis - to separate DNA fragments by size
Fig.T2.1
Small fragments migrate
more rapidly than large ones
Fig.T2.2
“Pulsed field” electrophoresis for separation of large DNA molecules
For example:
- restriction fragments generated by
“rare cutters”
- megaplasmids
- whole chromosomes (eg. yeast)
Fig.3.30
But if DNA is very complex, number of fragments (of various
sizes) generated is too large to see discrete bands after
electrophoresis...
so “continuum” of signals in lane
1 2
Why are different profiles expected for genomic DNA
cleaved with BamHI (6 bp cutter) vs. Sau3A (4 bp cutter)?
B
50 kb 20 kb 10 kb 5 kb 2 kb 1 kb -
S
Distance (on average) expected
between restriction sites
depends on probability of
occurrence of that sequence
Enzyme with 6 bp recognition site
expected to cut a DNA molecule (of
50% GC content) on average once
every 46 bp (ie. 4096 bp) (see p.86)
0.5 kb Lane 1 = uncut DNA
Lane 2 = 6 bp cutter
Lane 3 = 4 bp cutter
3
2. Southern hybridization
- to detect specific restriction fragment containing sequence
(eg.gene) of interest (vs. all other fragments)
Fig.2.11A
1. After electrophoresis, denature DNA and transfer it from gel to
membrane (eg. by capillary action or electroblotting…)
…so that DNA fragments remain in same relative positions
- probe will anneal with single-stranded DNA
on blot, if sequences are complementary
Fig.2.11B
2. Hybridize blot with “probe” (DNA, oligomer, cDNA, or RNA…
which is tagged either radioactively or non-radiolabelled)
and detect specific hybrid by autoradiography
Stability of hybrid depends on:
- length of hybrid, (eg for oligomer probes), GC content …
- hybridization conditions (such as temperature, ionic strength…)
Some applications of Southern blot analysis
- to identify restriction fragment
carrying sequence (eg gene) of interest
- to identify gene copy number (eg. multi-gene families)
Aside: Northern hybridization – RNA is electrophoresed, blotted
to membrane and hybridized with probe (eg gene of interest)
- to determine if gene is active, size of mRNA & its abundance …
(to be discussed in Topic 6)
(Fig.5.11)
3. PCR - polymerase chain reaction
- to obtain one specific DNA region in large copy number
- rapid amplification of DNA region
of interest by enzymatic reaction in
test tube
1. Denaturation of duplex DNA
2. Annealing of 2 different primers
(synthetic oligomers, usually 15-25 nt )
- flank region of interest,
- in opposite orientation
… so anneal to opposite strands of DNA
3. Extension of complementary strands
- cycle repeated 25-30 times
First cycle
Video at http://www.maxanim.com/genetics/PCR/pcr.swf
http://www.youtube.com/watch?v=x5yPkxCLads
Fig.2.28
"Scientists for Better PCR" a Bio-Rad Music Video for
the all new 1000-Series Thermal Cyclers”
Subsequent PCR cycles
- discrete PCR product generated
- its length corresponds to distance
between primers (including the
primers)
- sequences at ends of amplicon
correspond to the 2 primers used
Fig.2.29
Designing PCR primers:
5’
3’
3’
5’
5’
…GATTCC...
3’
…CTAAGG…
…GCGTAT...
…CGCATA...
3’
5’
Typically use 20-25 nt oligomers,
but for simplicity (as on a test) 6’mers are shown here
Why choose ~ 20’mers?
Tip: see Question 2.5 in text (p.61)
- for specificity…
How many times would a particular 20’mer sequence be expected
to be present in the human genome, by chance?
Why choose ~ 50% GC?
(and avoid homopolymeric stretches)
- to reduce chance of non-specific annealing at other genomic sites
How to double-check that PCR product (amplicon) is correct one?
1. Is it the right size?
- agarose gel electrophoresis (with size markers)
Well
Fig. 2.30
2. Does it contain the right sequence (eg gene X)?
- Southern hybridization - using gene X (eg. clone) as probe
- restriction analysis - are expected restriction sites present?
- nested PCR
New primer
- design “internal” primers
to use in 2d PCR experiment with
1st PCR product as template DNA
New primer
3’
5’
RT-PCR
3’
5’
3’
5’
3’
5’
3’
5’
Gene-specific oligomer
or oligo dT
5’
3’
5’
3’
5’
3’
- then sequence RT- PCR product
directly (or after cloning)
- (need sequence data to
design primers for RT-PCR)
5’
3’
3’
5’
(see p.142, Chapter 5)
Real-time quantitative RT-PCR
- detection and measurement of products generated during
each cycle of PCR by using a reporter fluorescent probe
eg. SYBR green, TaqMan
- to measure relative or absolute
amount of mRNA present in different
tissue types/developmental
stages/environmental conditions…
ΔRn : increment of fluorescent signal
at each time point
CT : PCR cycle number where reporter
fluorescence is greater than threshold
http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechQPCR.shtml
NCBI Technologies website
RFU = relative fluorescence units
NTC = no template control
Powers & pitfalls of PCR
- rapid method to generate large amounts of specific segment
of DNA (product usually < 10 kb in length)
- need very small amount of template DNA
…but can lead to contamination problems
- need prior sequence info to design primers
Some applications of PCR:
- genomic analysis
- RNA studies (RT-PCR)
- forensic work
- paleobiology (“ancient” DNA)
4. CLONING
- to obtain one specific DNA region in large copy number
- by using host cell (eg. E.coli) to amplify DNA of interest
- DNA fragments ligated
into vector
… then introduced into
bacterial (or yeast…) cell
by transformation
to generate clone library
= collection of clones
whose inserts cover the
entire genome
Fig. 2.15
Aside: cDNA library - mRNAs
reverse-transcribed into cDNAs
and cloned (Fig. 5.32)
Examples of cloning vectors used to generate clone library (or bank)
1. Plasmid
- to clone < 10 kb fragments
- origin of replication, selectable markers
Table 2.4
eg. antibiotic resistance in bacteria: ampicillin, tetracycline …
or nutrient requirement in yeast: URA3, TRP1 …
Insert disrupts lacZ’ gene,
so Xgal on plate not
converted to blue colour &
colonies are white
lacZ’ = marker for rapid
screening of recombinants
Fig.2.18
2. Phage lambda
- to clone 15-20 kb DNA fragments
(Aside: also l vectors for cloning cDNAs of 1-5 kb)
Mid-region of l DNA molecule can be removed
and replaced with similar-sized insert DNA of
interest, then packaged in phage particle
3. Cosmid
- l-plasmid hybrid, cos site to package DNA in phage particle
- to clone ~40-45 kb fragments
4. BAC
- bacterial artificial chromosome (~8 kb) with F (fertility) plasmid
origin of replication
- to clone ~ 300 kb fragments
- most commonly used vector for cloning large DNA fragments
5. YAC
- yeast artificial chromosome
- to clone ~ 1 Mbp fragments
- but sometimes DNA rearrangements & instability of inserts
Fig.2.25
Selectable markers (TRP1 & URAS3)
- yeast host strain requires tryptophan & uracil in medium to
grow, but transformants (which possess TRP1 & URA3 genes
on YAC) can grow in medium lacking them
How many clones needed in library to cover a
complete genome?
Depends on: genome size and insert size in vector
Number of clones N that must be screened to isolate
a given sequence with a probability of P:
N=
ln (1 – P)
)
ln (1 – insert length/ genome length)
Table 2.4
Rule-of-thumb:
For 99% probability of success, the total # bp present
in clones screened must be about 5 x greater than total genome size
For E.coli (genome size ~ 4.6 Mbp), how many clones
needed if average insert size = 10 kb?
Strategies to generate overlapping clones?
- use in assembling genomic maps
1. Use two clone banks with restriction fragments derived from different restriction
enzymes (or from incomplete digestion with one restriction enzyme)
A
…
A
B
A
B
B
A B A
A BA
B
DNA cleaved
with B
DNA cleaved
with A
A A
A
A
A
A
A
A
A
B B
A
A
B
B
etc.
prepare library
A
…
Can use clone from B library as
probe to find clone in A library
that contains part of the same
sequence…
B
B
B B
B
B
prepare library
B
plus neighbouring sequences…
A
A
Then can look for overlapping
clone in B library...
“chromosome walking”
etc.
B
B
B
2. Random fragmentation of DNA (eg sonication or nebulization )
then blunt-end ligation into vector (or ligation into vector
after linkers containing restriction sites added)
1 kb
Recover DNA of desired size
(eg. 1 kb) by gel electrophoresis
(or repeated nebulization) &
prepare clone library
Nebulizer for random
shearing of DNA
Cold Spring Harbor Protocols 2010
... having random
overlapping segments
of genome
Aside: this method was used to obtain the first complete bacterial
genome sequence (Haemophilus influenza) (Topic 5 & Fig.4.10)