NUCLEIC ACIDS. Amplification and Sequencing. A. Extraction and Isolation of Nucleic Acids. Cell lysis: rupture cell membrane and release content

NUCLEIC ACIDS Amplification and Sequencing A. Extraction and Isolation of Nucleic Acids • Cell lysis: rupture cell membrane and release content – tre...
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NUCLEIC ACIDS Amplification and Sequencing

A. Extraction and Isolation of Nucleic Acids • Cell lysis: rupture cell membrane and release content – treatment with hypotonic solution – treatment with surfactants (SDS or Triton X-100) – treatment with enzyme (lysozyme)

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CsCl density gradient centrifugation • Separates DNA from RNA and Proteins according to buoyant densities

1. Chromosomal DNA 2. Plasmid DNA

Total Cellular DNA Isolation • Cell culture is transferred to detergent solution (buffer containing SDS or Triton X-100) • Detergent disrupts cell walls and DNA-protein complexes • RNA is degraded with ribonuclease • Proteins can be digested by a proteolytic enzyme • DNA is extracted by precipitation with ethanol

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A protocol for Isolation of Plasmid DNA • Centrifuge cell suspension • Resuspend cell in cell lysis solution • Mix then centrifuge lysate cell debris are removed • Transfer clear lysate to resin mini-column • add ethanolic solution – Proteins and RNA elute

• Elute DNA by washing the column with water that is Nuclease free • Determine concentration by recording absorbance at 260 nm – 1AU corresponds to 50µg/ml (50 ng/µL)

• Store DNA at -20ºC

RNA Isolation-The Proteinase K Method •

Problems – –



RNA tends to form tight complexes with proteins Ribonuleases are ubiquitous

Proteinase K Method 1. Cell lysis with hypotonic solution 2. Separation of DNA and debris by centrifugation 3. Dissociation of RNA-Protein Complexes •

Treatment with proteinase K

4. Separation of digestion products •

Extraction by phenol or chlorofrom

5. Precipitation of RNA with ethanol

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B. Nucleic Acid Amplification – The Polymerase Chain Reaction (PCR) • PCR is an in vitro technique for reproduction/ amplification of DNA sequences by enzyme catalysis • Karl Mullis – Discovered PCR in 1983 – Was awarded the Nobel Prize in 1993

• Applications – – – –

Medical diagnosis Genetics Forensics Clinical medicine

Principle of PCR - Reagents • Excess of two primers: oligonucleotides that are complementary to the ends of the targeted nucleic acid region • DNA polymerase: catalyses addition of deoxynucleotide triphosphates (dNTPs) to growing chain along a complementary chain • Excess of the four deoxynucleotide triphosphates (dATP, dCTP, dGTP and dTTP) • Template DNA: DNA sample to be amplified • Buffer containing Mg2+

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PCR Principle – Amplification Reaction • Amplification reaction takes place in a thermocycler • Step 1- DNA denaturation – at 95ºC for 30 seconds – H-bonds are disrupted and double stranded DNA (dsDNA) is denatured to sinlge strand DNA (ssDNA) – First cycle denaturation is allowed during 2-5 minutes • Step 2- Primer annealing: at 5060 ºC for 30 seconds – Primers anneal with the complementary region of the target DNA

PCR Principle – Amplification Reaction • Step 3- Primer extension (60 seconds) – Synthesis of DNA is catalyzed by DNA polymerase at 72ºC – Primer extension proceeds from the 3’-end to the 5’end of the template – 3’end of the hybridized primer is extended along the original DNA strand by addition of nucleotides to the chain – Last extension step can be extended (~5 minutes) • 20 to 30 cycles are performed

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Example of a set of Experimental conditions for PCR • Concentration of primer – 50 ng/µL = 50 µg/mL

• Concentration of dNTPS – 10mM

PCR Principles - Results • Theoretical yield

N m = N0 2n N : number − of − molecules n : number − of − cycles

• Limiting factors ¾ Depletion of reagents ¾ Amplification of longer strands during the first cycles ¾ Reaction efficiency: • Empirical yield :

N m = N 0 (1 + x )n x : reaction − efficiency(0 → 1)

• High specificity- only target sequence produced • Fidelity – minimum polymerase induced errors

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Rate of Amplification During PCR • Amplification of target section of DNA proceeds according to three distinct phase. – Starting phase• first cycle- copying from each end results in the synthesis of two types of copy of longer length than target • Second cycle- first copies of single stands target are produced • Third- first copies of double strands of the target

– Phase of exponential growth• Forth cycle: geometric increase starts

– A plateau • • • •

Due to depletion of reagent Deterioration of DNA polymerase and inhibition of DNA polymerase by released phosphate Hybridization of longer strands instead of hybridization of ssDNA and primer

Rate of Amplification During PCR

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DNA Polymerase • Must be heat stable • Must have good polymerisation activity: i.e. able to synthesize long stretches of DNA • Pfu polymerase – from hyperthermophilic archeon Pyrococcus furiosis – possesses 5’-3’ exonuclease activity which improves fidelity of replication – Incorrect nucleotides are recognized, removed and replaced

Primers • Short oligonucleotides complementary to the ends of the target sequence • Length: 10 – 30 nucleotides • Two primers – Forward primer – Reverse primer

• Complementary sequence must be unique in the template • No intra of inter primer complementarity to prevent formation of primer dimers • Ideally equal number of each base • Avoid long stretches of repetitive sequences • Melting temperature for both primers should be similar a should lie between 55 and 80°C

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dNTPs, Buffer, MgCl2, BSA • Concentration of dNTPs should be equal • Buffer pH, ionic strength adjusted to optimize the catalytic efficiency of the polymerase used • Ionic strength has a crucial influence on the specificity of the PCR – Typically 50 mM Tris-HCl, pH 8.3 with KCl of NaCl

• MgCl2: 0.5 and 5mM:

– Mg2+ forms a soluble complex with DNA and polymerase thus bringing them in close proximity – Mg2+ balances the charges on DNA. – Mg2+ is a polymerase effector – At low Mg2+ concentration activity of poly is decreased – high concentration of Mg2+ increase annealing of the primers to incorrect sites

• BSA: added to stabilize polymerase

Real-Time PCR • Monitoring PCR product production • Quantitative PCR (QT-PCR) • DsDNA binding assay used to monitor product – Dye is used that fluoresces upon binding to dsDNA – Intercalator dyes • Ethidium bromide • Daunomycin • Actinomycin – Minor grooves binders : Distamycin, Netrospin, 4,6diamidino-2-phenylindole

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QT-PCR with Intercalator Dye

QT-PCR Probe-based Assay • Rely on degradation of a target fluorescent probe by 5’-3’ exonulease activity of the polymerase • Probe is a oligonucleotide complementary to a region of the target between two primers • Fluorescent reporter is attached to the probe at the 5’-end • A quencher is attached to the 3’-end • Probe is added to the PCR mixture and hybridizes to the ssDNA after denaturation • Due to proximity of R and Q, fluorescence is reduced

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QT-PCR Probe-based Assay • During extension reporter is cleaved due to exonuclease activity and fluoresces • Fluorescence is proportional to number of cycles

Reverse Transcriptase-PCR (RT-PCR • Used when RNA is available – E.g. HIV virus

• The enzyme RNA-directed DNA polymerase ( reverse transcriptase) is used – Transcribes mRNA to its complementary strand of DNA

• RNA template is denatured at 72ºC • RNA is cooled to 42ºC – Primers anneal

• Reverse transcriptase catalyses the extension of primers in the 5’ to 3’ direction generating cDNA • Reaction is heated to 94ºC: reverse transcriptase is inactivated • PCR reagents are added for cDNA amplification

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C. Nucleic Acid Sequencing • Chemical Cleavage Method • Chain Terminator Method (Sanger or Dideoxy Method) • Walter Gilbert and Frederic Sanger were awarded the Noble Prize in 1980 for their pioneer work

C.1 Use of restriction enzyme in Sequencing • Reduce native DNA to smaller fragments of approximately 800 base pairs – Restriction endonucleases recognize a specific base sequence of 4 to 8 bases within the dsDNA and cleave both strands at a point close to this site – BamHI (Bacillus amyloliquefacciens H) G♦GATCT – XhoI (Xanthomonas holcicola) C♦TCGAG – SalI (Streptomyces albus G) G♦TCGAC – TaqI (Thermus aquaticus) T♦CGA (N6-Methlyladenine)

• • • •

Denature into ssDNA (melt dsDNA) Separate ssDNA fragments by GE Repeat digestion if there is DNA that is longer than 800 base pairs Determine length of ssDNA products by comparing to standards (restriction map)

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Note on Restriction Enzyme and Design of DNA template

C.2 Chemical Cleavage (Maxam-Gilbert) Method • ssDNA is labelled at the 5’-end with a 32P atom – React ssDNA with [γ32P]ATP in the presence of polynucleotide kinase – 5’-phosphate is removed first with alkaline phosphatase

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C.2 Chemical Cleavage/ Maxam-Gilbert Method) • Labeled DNA is treated with a reagent that cleaves DNA at a particular type of nucleotide • Hydrazine cleaves DNA before every C-nucleotide at 1.5 M NaCl • Reaction must have low yield so as to obtain random distribution of different length due to cuts at all the sites

• Labeled fragments are separated by SDS-PAGE

Cleavage reaction for G residues • When residues are protonated, cleavage occurs before both G and A

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Example of cleavage before C • Labeled DNA to be sequenced – 32P-ACCTGACATCG

• Cleavage products – 32P-ACCTGACAT – 32P-ACCTGA – 32P-AC – 32P-A

Reagents for cleaving DNA • Aliquot 1 Cleavage at only G – DNA treated with Dimethyl sulfate (DMS) – Methylation of G residues at the N7 position – the glycoside bond of the methylated G residue is hydrolyzed and the G residue is eliminated. – Piperidine is added which reacts with hydrolyzed sugar residue, cleavage of the backbone results

• Aliquot 2 cleavage at G and A – Use acid instead of DMS – Position of A revealed

• Aliquot 3: cleavage at C and T – Treat with hydrazine, then piperidine

• Aliquot 4: cleavage at C only – Treat with hydrazine in the presence of 1.5 M NaCl – Position of T revealed

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Maxam Gilbert Method

C.3 The Chain Terminator Method (The Sanger or Dideoxy) • Synthesize complementary DNA like in PCR, but in the presence of a chain terminating nucleotide • Four aliquots each incubated with DNA polymerase, four dNTPs and a suitable primer • α-32P is incorporated in primer. This labels the complementary strands for analysis • A small amount of one of the 2’,3’-dideoxynucleotide triphosphate (ddNTP) is added – Incorporation of ddNTP terminates the reaction as there is no free 3’-hydroxyl group

• Products are separated in parallel according to size by GE and sequence is determined from the autoradiogram

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Sanger Method

Automated

Commercial Sanger • Strategy I – Four different reaction mixtures are set up – Primers covalently bonded to fluorescing dye at the 5’-terminus – Reaction products are separated by gel electrophoresis in parallel lanes – Laser induced fluorescence (LIF) is detected

• Strategy II – Primers in each reaction mixture are labeled with different fluorescent dyes – Reaction mixtures are mixed at the end of the reaction and separated in a single lane by gel electrophoresis – The terminal base of each fragment is identified by the fluorescence of the dye on the associated primer

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Commercial Sanger • Strategy III – A single vessel used – Each of the ddNTPs is covalently bonded to a different fluorescent dye – The products are separated in single lane – The terminal base is identified by the characteristic fluorescence of the dye attached to the terminator

Examples of data expected when ddTTP and ddCTP are the chain terminators

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Different fluorescent labels on each type of terminator Univerisity of Michigan DNA Sequencing Core

Automated Sequencing Gel

http://seqcore.brcf.med.umichi.edu/doc/dnapr/sequencing.html

• Date from sequencer computer – Colors detected in one lane (one sample) – scanned from the smallest to the largest

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State-of-the-Art “Sanger Method” Instrument High throughput Computerized procedures Robotics Short analysis time afforded by CE

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