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