DNA and RNA isolation and purification (course readings 10 and 11)

I. Genomic DNA preparation overview

II. Plasmid DNA preparation

III. DNA purification• Phenol extraction• Ethanol precipitation

IV. RNA work

What do we need DNA for?

•Detect, enumerate, clone genes•Detect, enumerate species•Detect/sequence specific DNA regions•Create new DNA “constructs” (recombinant DNA)

What about RNA?

•Which genes are being transcribed?•When/where are genes being transcribed?•What is the level of transcription?

cell growthcell harvest and lysis

DNA purification

DNA purification: overview

DNA concentration

Bacterial genomic DNA prep: cell extract

Lysis:

• Detergents• Organic solvent• Proteases (lysozyme)• Heat

“cell extract”

Genomic DNA prep: removing proteins and RNA

Add the enzyme RNase to degrade RNA in the aqueous layer

Need to mix gently! (to avoid shearing breakage of the genomic DNA)

chloroform

2 ways to concentrate the genomic DNA

70% final conc.

“spooling” Ethanol precipitation

Genomic DNA prep in plants -- how get rid of carbohydrates?

CTAB:

Cationic detergent

(MC 6.61-6.62)

(low ionic conditions)

N+

CH3

Br-

CH3

CH3C16H33

Plasmids: vehicles of recombinant DNA

Bacterial cell

genomic DNA plasmids

Non-chromosomal DNAReplication: independent of the chromosomeMany copies per cellEasy to isolateEasy to manipulate

Plasmid purification: alkaline lysis

Alkaline conditions denature DNA

Neutralize: genomic DNA can’t renature (plasmids CAN because they never fully separate)

DNA purification: silica binding

Binding occurs in presence of high salt concentration, and is disrupted by elution with water

DNA purification: phenol/chloroform extraction

1:1 phenol : chloroformor

25:24:1 phenol : chloroform : isoamyl alcohol

Phenol: denatures proteins, precipitates form at interface between aqueous and organic layer

Chloroform: increases density of organic layer

Isoamyl alcohol: prevents foaming

1. Aqueous volume (at least 200 microliters)

2. Add 2 volumes of phenol:chloroform, mix well

3. Spin in centrifuge, move aqueous phase to a new

tube

4. Repeat steps 2 and 3 until there is no precipitate

at phase interface

5. (extract aqueous layer with 2 volumes of

chloroform)

Phenol extraction

Ethanol depletes the hydration shell surrounding DNA…

• Allowing cations to interact with the DNA phosphates

• Reducing repulsive forces between DNA strands

• Causing aggregation and precipitation of DNA

• Aqueous volume (example: 200 microliters)

-- add 22 microliters sodium acetate 3M pH 5.2

-- add 1 microliter of glycogen (gives a visible pellet)

-- add 2 volumes (446 microliters) 100% ethanol

-- mix well, centrifuge at high speed, decant liquid

-- wash pellet (70% ethanol), dry pellet, dissolve in

appropriate volume (then determine DNA

concentration)

Ethanol precipitation (DNA concentration)

cell growthcell harvest and lysis

DNA purification

DNA purification: overview

DNA concentration

Isolation of RNA -- Course reading 11

DNA --------------> mRNA --------------> protein

Lots of information in mRNA:

When is gene expressed?What is timing of gene expression?What is the level of gene expression?

(but what does an mRNA measurement really say about expression of the protein?)

RNA in a typical eukaryotic cell:

10-5 micrograms RNA

80-85% is ribosomal RNA15-20% is small RNA (tRNA, small nuclear RNAs)

About 1-5% is mRNA

-- variable in size-- but usually containing 3’ polyadenylation

The problem(s) with RNA:

RNA is chemically unstable -- spontaneous cleavage of phosphodiester backbone via intramolecular transesterification

RNA is susceptible to nearly ubiquitous RNA-degrading enzymes (RNases)

RNases are released upon cell lysisRNases are present on the skinRNases are very difficult to inactivate

-- disulfide bridges conferring stability-- no requirement for divalent cations for

activity

Common sources of RNase and how to avoid them

Contaminated solutions/buffers

USE GOOD STERILE TECHNIQUETREAT SOLUTIONS WITH DEPC (when possible)MAKE SMALL BATCHES OF SOLUTIONS

Contaminated equipment

USE “RNA-ONLY” PIPETS, GLASSWARE, GEL RIGSBAKE GLASSWARE, 300°C, 4 hoursUSE “RNase-free” PIPET TIPSTREAT EQUIPMENT WITH DEPC

Top 10 sources of RNAse contamination(Ambion Scientific website)

1) Ungloved hands2) Tips and tubes3) Water and buffers4) Lab surfaces5) Endogenous cellular RNAses6) RNA samples7) Plasmid preps8) RNA storage (slow action of small amounts of RNAse9) Chemical nucleases (Mg++, Ca++ at 80°C for 5’ +)10) Enzyme preparations

Inhibitors of Rnase

DEPC: diethylpyrocarbonate

alkylating agent, modifying proteins and nucleic acids

fill glassware with 0.1% DEPC, let stand overnight at room temp

solutions may be treated with DEPC -- add DEPC to 0.1%, then autoclave (DEPC breaks down to CO2 and ethanol)

Inhibitors of Rnase

Vanadyl ribonucleoside complexescompetitive inhibitors of RNAses, but need to be

removed from the final preparation of RNA

Protein inhibitors of RNAsehorseshoe-shaped, leucine rich protein, found in

cytoplasm of most mammalian tissuesmust be replenished following phenol extraction

steps

Making and using mRNA (1)

Making and using mRNA (2)

Purifying RNA: the key is speed

Break the cells/solubilize components/inactivate RNAses by the addition of guanidinium thiocyanate (very powerful denaturant)

Extract RNA using phenol/chloroform (at low pH)

Precipitate the RNA using ethanol/LiCl

Store RNA:in DEPC-treated H20 (-80°C)in formamide (deionized) at -20°C

Selective capture of mRNA: oligo dT-cellulose

Oligo dT is linked to cellulose matrix

RNA is washed through matrix at high salt concentration

Non-polyadenylated RNAs are washed through

polyA RNA is removed under low-salt conditions

(not all of the non-polyadenylated RNA gets removed

Other methods to capture mRNA

Poly(U) sepharose chromatography

Poly(U)-coated paper filters

Streptavidin beads:

•A biotinylated oligo dT is added to guanidinium-treated cells, and it anneals to the polyA tail of mRNAs

•Biotin/streptavidin interactions permit isolation of the mRNA/oligo dT complexes

How good is the RNA prep?

The rRNA should form 2 sharp bands in ethidium bromide-stained gels (but mRNA will not be visible

Use radiolabelled poly dT in a pilot Northern hybridization--should get a smear from 0.6 to 5 kb on the blot

Use a known, “standard” gene probe (e.g. GAPDH in mammalian cells) in Northern hybridization--there should be a sharp band with no degradation products

In vitro amplification of DNA by PCR

I. Theory of PCRII. Components of the PCR reactionIII. A few advanced applications of PCR

a) Reverse transcription PCR (for RNA measurements)

b) Quantitative real-time PCRc) PCR of long DNA fragmentsd) Inverse PCRe) MOPAC (mixed oligonucleotide priming)

Molecular Cloning, p. 8.1-8.24

What is PCR?

• Polymerase Chain Reaction--first described in 1971 by Kleppe and Khorana, re-described and first successful use in 1985

• Allows massive amplification of specific sequences that have defined endpoints

• Fast, powerful, adaptable, and simple*

• Many many many applications

* usually

Why amplify specific sequences?

• To obtain material for cloning and sequencing, or for in vitro studies

• To verify the identity of engineered DNA constructs

• To monitor gene expression• To diagnose a genetic disease• To reveal the presence of a micro-

organism• To identify an individual• Etcetera, etcetera

What you need for PCR:

1. Template DNA that contains the “target sequence”

2. Primers: short oligonucleotides that define the ends of the target sequence

3. Thermostable DNA polymerase

4. Buffer, dNTPs

5. A thermal cycler

Denaturation: denature template strands (94°C for2-5 minutes), can also add your DNA polymerase atthis temp. for a “hot start” (adding DNA pol to a hottube can prevent false priming in the initial round ofDNA replication)

Annealing: The default is usually 55°C. Thistemperature variable is the most critical one forgetting a successful PCR reaction. This is the bestvariable to start with when trying to optimize a PCRreaction for a specific set of primers. Annealingtemperatures can be dropped as low as 40-45°C,but non-specific annealing can be a problem

A typical PCR program:

Extension: generally 72°C, this is the operatingtemperature for many thermostable DNApolymerases.

Number of cycles: Depends on the number ofcopies of template DNA and the desired amountof PCR product. Generally 20-30 cycles issufficient.

A typical PCR program:

How it works:

a simple PCR reaction, first cycle

(Can also be Single-stranded)94°C

50°C

72°C

Cycles ofdenaturation,primer annealing,and primerextension by DNApolymerase

a simple PCR reaction, second cycle

like firstcycle

newreactions

a simple PCR reaction, third cycle

PCR animation:http://www.dnai.org/b/index.htmlhttp://www.dnalc.org/ddnalc/resources/shockwave/pcranwhole.html

Choosing primers:• Should be 18-25 (17-30?) nucleotides in length (giving specificity)

• Calculated melting temperature varies depending on the method used (55-65°C using the Wallace Rule, eg. see MC), but should be nearly identical for both primers

• Avoid inverted repeat sequences and self-complementary sequences in the primers, avoid complementarity between primers (‘primer dimers’)

• Have a G or C at the 3’ end (a G/C “clamp”)

• Many computer programs exist for helping meet these criteria (ex: Biology Workbench, workbench.sdsc.edu)

Thermostable DNA polymerases

• Isolated from thermophilic bacteria and archaea (T. aquaticus is a bacterium, not an archaeon)

• Bacterial enzymes (e.g. Taq) good for routine reactions and small PCR products, fidelity of replication is somewhat low

• Archaeal enzymes (e.g. Pfu) also good for routine reactions and best for cloning: 3’--5’ exonuclease activity provides very high fidelity, and enzymes are very stable to heat

(See Molecular Cloning table 8-1)

Thermal cyclersStandard: heat block, “ramp” times fairly long (10 -20 seconds to change temperature), 30 cycle PCR lasts 2-3 hours.

Advantage: easily automated, heat blocks can PCR up to 384 samples at a time

Disadvantage: relatively slow

New: reactions are being sped up significantly

--capillary tubes heated and cooled by blasts of air--30 cycle-PCR done in >30 minutes (harder to scale up)

--fluid flow cells: channels force liquid through temperature gradients, very fast (but still not widely available)

Sources of problems in PCR • Inhibitors of the reaction from the the

template DNA preparation (protease, phenol, EDTA, etc)

• Cross-contamination by DNA from sources other than the template added– if this becomes a problem:

• Work in a laminar flow hood (decontaminate using UV light 254 nm)

• Use PCR dedicated pipettors (with barrier tips), PCR dedicated reagents

• Centrifuge tubes before opening them to prevent spattering, pipet contamination

Controls to include in difficult PCRs:

BystanderDNA

templateDNA

TargetDNA

Specificprimers

Positivecontrols

1 + - + +

2 - - + +Negativecontrols

3 - - - +

4 + - - +

Bystander DNA: not recognized by primers

Target DNA: known to contain primer recognition sequences

Hot Start of PCR reactions

• Witholding some component of the reaction until the denaturing temperature is reached (94°C)

• This helps prevent non-specific priming, which may occur at the low temperatures (room temp.) -- the non-specific priming could give artifactual amplification as PCR block temperature rises

A) Wait until 94°C to add enzyme --or--B) Use enzyme bound to an inactivating enzyme

antibody that releases at high temperature --or--

C) Use wax beads containing Mg++ that can only be released at high temp.

Touchdown PCR

• Useful if your primers are not 100% complementary to your template DNA (e.g. degenerate oligos), or when there are multiple members of the gene family you are amplifying• Allows you to selectively amplify only the best sequences (with the least mismatches) while minimizing non-specific PCR products

• Start with 2 cycles at an annealing temperature about 3°C higher than the calculated primer melting temperatures.• Progressively reduce the annealing temperature by 1°C at 2 cycle intervals

Trouble-shooting:

(see Molecular cloning, tables 8-4 and 8-5)

-- Very little product-- No PCR product-- Multiple bandsEtc.

III. Special applications for PCR

A. Reverse transcription PCR (for RNA measurements)

B. Quantitative (real-time) PCR

C. PCR of long DNA fragments

D. Whole genome amplification

E. Inverse PCR

E. MOPAC (mixed oligonucleotide priming)

Amplification of RNA (monitor gene expression): reverse transcription PCR (RT-

PCR)Step 1: generate a 1st strand cDNA using reverse transcriptase (catalyzes synthesis of DNA from an RNA template)

Step 2: normal PCR (from cDNA) using gene-specific primers

A)

B)

C)

Quantitative(real time)

PCRThe more target DNA there is, the more probe anneals, the more it is cleaved (by Taq’s 5’-3’ exonuclease activity)

Fluorescence measurements are done simultaneously with PCR cycles, yields an instantaneous measurement of product levels

Quantitative Real Time (QRT) PCR

Position of the steep part of the curve varies depending on the amount of template DNA or RNA, can measure variation over 5 or 6 orders of magnitude

moretemplate less

template

Another quantitative measure of double stranded DNA in a PCR reaction: binding of SYBR Green Dye

From the Molecular Probes website (www.probes.com)

Non-fluorescing SYBR green dye

Fluorescing SYBR green dye

Use of a quenching dye to reduce measurement of “primer dimer” artifacts in QRT-PCR

QSY quencher dye: it absorbs fluorescence from sybr green dyes in the vicinity--prevents accumulation of signal from primer dimers

Always do your controls! QRT-PCR using Sybr green dye fluorescence

(From the Invitrogen website)

Standard curve: what is “threshold” for specific number of DNA molecules?

PCR of long sequences (>2 kb)

Long DNAs are difficult to amplify– Breakage of the DNA– Non-processive behavior of DNA

polymerase– Misincorporation by error prone DNA

polymerases

Changes to protocol that assist in long PCR – Make sure DNA is exceedingly clean– Use DNA polymerase “cocktail”: Taq

for it’s high activity, and Pfu for its proofreading activity (it can actually correct Taq’s mistakes)

– Increase time of extension reaction (5-20 minutes, compared to the standard 1 minute for short PCRs)

PCR of long sequences (>2 kb)

•Amplified product longer than 3 kilobases with high fidelity•10 times fewer mutations than with conventional PCR

•Taq DNA pol (no proofreading) plus an archaeal DNA pol (does proofreading)

•Betaine (amino acid analogue with several small tetraalkyammonium ions)--reduces non-specific amplification products--reduces non-complementary primer-template interactions? (unknown how it works)

Whole genome amplification: multiple displacement amplification (MDA)

Applications: forensics, embryonic disease diagnosis, microbial diversity surveys, etc.

How it works: Strand-displacement amplification used by rolling-circle replication systems.

Phi29 DNA polymerase (very low error rate) and random hexamer primers, low temperature! (30°C)

Whole genome amplification : multiple displacement amplification (MDA)

20-30 micrograms human DNA can be recovered from 1-10 copies of the human genome

Distribution of products appears to be random sampling of the available template (and this is good!)

Inverse PCR: sequencing “out” from

known sequence

“Vectorette” PCR

First primer: known sequenceVectorette primer: only in vectorette-ligated sequence--it cannot anneal until there is a single round of primer extension from the specific primer

http://www.bio.psu.edu/People/Faculty/Akashi/vectPCR.html

MOPAC: Mixed oligonucleotide primed amplification of cDNA

If you only have a protein sequence, and you want to clone the gene for the protein:

1. Design oligonucleotides based on deduced mRNA (and DNA) sequence (but since multiple codons can encode the same amino acid, this gets complicated quickly)

2. program your oligo synthesizer to make primer sets that are randomized for the degenerate positions of each codon

3. use universal nucleotides like inosine, which base pairs with C, T, and A (limits degeneracy)

4. Do your PCR and hope for the best