TECHNIQUES & TOOLS FOR STUDYING DNA

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)