January 19, 2016 Biotech 3 Lecture 8

2. Annealing 1. Melting 3. Elongation 4. Repeat cycle ~ 30 times Polymerase Chain Reaction

Inserting a DNA fragment into a plasmid

Cloning: vector plus insert

Cloning : vector + insert (with restriction sites)

Polymerase Chain Reaction Primers: restriction sites, tags, etc.

Cloning: vector plus insert with restriction sites

ComponentCommon ConcentrationFinal Concentration* DNA TemplateN/A50 – 500 ng genomic DNA** 50 pg – 50 ng plasmid DNA dNTPs10 mM0.2 mM PCR Buffer10X1X Forward Primer100 μM0.1 – 1 μM Reverse Primer100 μM0.1 – 1 μM DNA PolymeraseVaries depending on polymerase and manufacturer < 1 μl MgCl 2 *** 1.5 – 4 mM DMSO**** *Master mix ** Why is genomic DNA a higher amount? ***too low – no reaction, too high –nonspecific ****Optional Components in a PCR Reaction

Deoxyribonucleic acid

dNTPs Deoxyribonucleic acid Structure

1. Real-Time PCR (Quantitative PCR, qPCR) -Allows for quantitation of the amount of initial DNA in a sample -Quickly measure DNA and gene expression levels -Fluorescent stain binds to dsDNA -Compare fluorescence of known concentration of standards with PCR reaction Lower concentration Higher concentration Cq Type of PCR Reactions - qPCR

2. Reverse Transcription PCR (RT-PCR) -Uses retroviral reverse transcriptase to reverse transcribe mRNA into DNA before actual PCR begins -Resulting DNA is called complementary DNA (cDNA) -The cDNA is used as a template for PCR -Initial 42 °C hold step to allow reverse transcriptase to reverse transcribe mRNA into dsDNA -RT-PCR is used with real-time PCR (qPCR) to measure gene expression levels Type of PCR Reactions – RT-PCR

3. DNA Sequencing! (Kind of PCR, though not really, only use one primer) -Use 2’, 3’-dideoxynucleotides -ddNTPs along with dNTPs -Elongation terminates when a ddNTP is added to the growing fragment -Various size products are synthesized, each terminating at a single detectable nucleotide -Developed by Fred Sanger Type of PCR Reactions - Sequencing

Sanger DNA Sequencing

CycleStepTemp.DurationCycles Initial denaturation Denature DNA 94 °C5 min1x Thermal Cycling Denature94 °C1 min X Anneal primers 52 °C1 min Extend72 °C2 min Final extension Extend72 °C10 min1X Hold 4 °C1x PCR Program

1.Quantity of template DNA 2.Primer design 3.Annealing temperature 4.Cycling parameters: Cycles and extension time 5.Magnesium concentration PCR Program Variables

4.Cycling parameters: -Length of time of extension is dependent on DNA polymerase and size of target sequence PCR Cycling Parameters

Fidelity: Proof reading ability (3’ – 5’ exonuclease activity) Extension rate – kb/min or nt/sec (ex 1 kb/min) Product size A at the 3’ end – TOPO Cloning Optimal temperature – 70 to 75 °C Hot start polymerases – antibody binds to active site to prevent extension at lower °C Purity – higher purity required for low copy templates Quantity – amplification will plateau if too little is used Uses – routine PCR, colony PCR, short fragment, TA cloning, long PCR, complex templates, qPCR, high fidelity PCR EnzymeTaq Pol Fidelity8.9E -5 to 1.1E -4 Extension rate35 – 150 nt/sec A at 3’ end+ Product size<5 kb Proof read- Temp70 – 75 °C UsesRoutine PCR DNA Polymerase Enzyme Details

Front Cover

TOPO Cloning

TOPO Cloning – T overhangs

TOPO Cloning – Blunt end

TOPO Cloning – Directional