PCR & Electrophoresis Lecture Notes PDF

Biotechniques (BMS 34010A) Fall semester 2023 -2024 Dr. Tania Tahtouh [email protected] PCR & Electrophoresis  Learning outcomes  Explain PCR technique.  Identify some Polymerases.  Describe TA cloning.  Describe Gel electrophoresis.  Polymerase Chain Reaction (PCR) ...

Biotechniques (BMS 34010A) Fall semester 2023 -2024 Dr. Tania Tahtouh [email protected] PCR & Electrophoresis  Learning outcomes  Explain PCR technique.  Identify some Polymerases.  Describe TA cloning.  Describe Gel electrophoresis.  Polymerase Chain Reaction (PCR)  Polymerase chain reaction (PCR) is a laboratory technique used to amplify DNA sequences. ▪ The method involves using short DNA sequences called primers to select the portion of the genome to be amplified. ▪ PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA complementary to the offered template strand. ▪ The technique can produce a billion copies of the target sequence in just a few hours.  Polymerase Chain Reaction (PCR)  DNA-dependent DNA polymerase copies DNA into DNA DNA polymerase ▪ adds dNTP to a 3’OH end of an existing strand. 3 DNA-dependent DNA polymerase Reverse transcriptase RNA-dependent DNA-dependent 1 4 DNA polymerase RNA polymerase RNA-dependent 2 RNA polymerase RNA polymerase RNA replicase Components of PCR  DNA template - the sample DNA that contains the target sequence.  Primers - short pieces of single-stranded DNA that are complementary to the target sequence. The polymerase begins synthesizing new DNA.  Nucleotides (dNTPs or deoxynucleotide triphosphates) - single units of the bases A, T, G, and C, which are essentially "building blocks" for new DNA strands.  DNA polymerase - type of enzyme that synthesizes new strands of DNA complementary to the target sequence.  DNA polymerase buffer - a buffer that creates optimal conditions for the polymerase to work.  Water to adjust final concentrations.  Components of PCR  DNA template: 104-107 molecules (50-100 ng of gDNA OR 10-50ng of plasmid DNA).  Primers: 0.1–1 μM (degenerated primers 0.3–1 μM).  Nucleotides: 50 μM of each of the four nucleotides (20-200 μM).  DNA polymerase: 0.5-2.5 units per 50 μl.  DNA polymerase buffer - X buffer.  Sterile water to adjust final folume. Total volume: 15μL  Why are 2 primers needed for PCR?  Two primers, forward primer and reverse primer are designed to flank the target region for amplification. ▪ The forward primer binds to the template DNA. ▪ The reverse primer binds to the other complementary (coding) strand. Plus “+” Sense Coding Complementary Non-template Template Complementary Minus “-” Antisense Non-coding Orientation of primers  PCR primer design  Primers should generally have the following  Primers should g enerally have the following properties: properties: ▪ Length of 18-24 bases ▪ Length of 18-30 bases ▪ 40-60% G/C content ▪ 40-60% G/C content ▪ End with G/C (GC Clamp). ▪ Start and end with 1-2 G/C pairs ▪ Melting temperature (Tm) of 65-75°C ▪ Melting temperature (Tm) of 50-60°C ▪ Primer pairs should have a Tm within 5°C of each other ▪ Primer pairs should have a Tm within 5°C of each other ▪ Avoid regions of secondary structure, GC-rich and AT- ▪ Primer pairs should not have complementary regions rich domains. ▪ Avoid runs of 4 or more of one base, or dinucleotide repeats, e.g. ACCCC or ATATATAT  Note: If you will be including a restriction site at the 5’ end of your primer, note that a 3-6 bp "clamp" should be added upstream in order for the enzyme to cleave efficiently. PCR principle  PCR is based on three simple steps required for any DNA synthesis reaction: (1)denaturation of the template into single strands (2)annealing of primers to each original strand for new strand synthesis (3) extension of the new DNA strands from the primers. PCR profile At its optimal temperature (72°C), Taq polymerase incorporates nucleotides at a rate of 1-2 kb per minute. PCR program  Polymerases commercially available  DNA polymerase I. ▪ The commercial form is extracted from E. coli. ▪ Its 5' to 3' DNA polymerase activity requires a template. ▪ it also has 3' to 5' and 5' to 3' exonuclease activity. ▪ This enzyme is used to synthesize DNA from a single-strand DNA template at 37ºC.  The Klenow Fragment of DNA polymerase I. ▪ It is a fragment of DNA polymerase I obtained by limited proteolysis. ▪ The 5' to 3' exonuclease activity is removed. ▪ The 5' to 3' polymerase and the 3' to 5’ exonuclease activities are preserved.  Polymerases commercially available  Taq DNA Polymerase. ▪ This is a thermostable enzyme isolated from Thermus aquaticus. ▪ It is used for PCR amplification of DNA fragments up to 5 kb in length. ▪ It is also used for DNA labelling and sequencing. ▪ It is ideal for TA cloning. It has a non-template dependent activity that adds a single adenosine to the 3' ends. The downside of Pfu is its speed  Pfu DNA polymerase. which is slower than that of Taq. ▪ It derives from the hyperthermophilic archae Pyrococcus furiosus. ▪ It has 3' to 5' exonuclease activity and a high proofreading efficiency ▪ It lacks 5' to 3' exonuclease activity. ▪ It is used for high-fidelity PCR and primer-extension reactions and the generation of blunt-end amplification products.  Polymerases commercially available Commercially purified Taq polymerase At its optimal  Phusion. doesn't have a proof-reading domain, so temperature (72°C), Taq it has a higher error rate. polymerase i ncorporates ▪ It has extreme fidelity and high speed. nucleotides at a rate of ▪ It allows high product yields with minimal enzyme concentrations. 1-2 kilobases per minute. ▪ It is capable of amplifying long templates. Phusion requires 15-30  Terminal transferase. seconds per kb. This ▪ This is a mammalian enzyme expressed in lymphocytes. means 4 kb per minute. ▪ It catalyzes deoxynucleotide addition to a free 3'-OH end without the need for a template. The choice of deoxynucleotide added is made randomly. The base composition of the synthesized polydeoxynucleotide depends on the base concentrations in the incubation medium. ▪ It is an example of a DNA polymerase that does not require a primer. ▪ It is used to generate DNA blunt ends and for labelling of DNA 3' ends TA cloning  Taq polymerase has non-template dependent activity which preferentially adds a single adenosine to the 3'-ends of a double stranded DNA molecule, and thus most of the molecules PCR amplified by Taq polymerase possess single 3'-A overhangs.  TA cloning vector was designed so that when linearized, it has single 5′-T overhangs at each end.  TA cloning is one of the simplest and most efficient methods for the cloning of PCR products.  Gel electrophoresis  Gel electrophoresis is a laboratory method used to separate mixtures of molecules (DNA, RNA, or proteins) according to their molecular size. ▪ DNA is negatively charged. When an electric current is applied to the gel, DNA will migrate towards the positively charged electrode. ▪ The molecules to be separated are pushed by an electrical field through a gel that contains small pores. DNA is negatively charged  DNA is negatively charged because of the presence of phosphate groups in nucleotides.  The phosphate backbone of DNA is negatively charged, which is due to the presence of bonds created between the phosphorus and oxygen atoms.  Each phosphate group contains one negatively charged oxygen atom.  Agarose gel  Agarose is a group of natural polysaccharides derived from seaweed (red algae). It’s a derivative of agar.  Agarose can be dissolved in boiling water and a gel is formed after cooling this solution below 45 °C as a result of extensive hydrogen- bonding between the agarose chains.  The pore size of agarose gels depends on the agarose content.  6 % agarose have an average pore size of approximately 30 nm, whereas 4 and 2 % agarose beads have a pore size of 70 and 150 nm, respectively.  Agarose with even larger diffusion pores are used in gel electrophoresis to allow the passage of very large DNA molecules.  Polyacrylamide gels  3 major differences between agarose & polyacrylamide gels, which lead to their distinct uses in the research lab. ▪ Toxicity: agarose is considered entirely non-toxic, whereas polyacrylamide powders and gels are considered moderately hazardous and require protection during handling. ▪ Molecular complexity: Agarose is complex and has wide gaps between the many differently-sized molecules that make up the gel matrix. Polyacrylamide is made up of only one large molecular type. ▪ Gel preparation: Agarose is poured horizontally, and polyacrylamide is poured vertically.  degrading the nucleic acids. Running buffers  The function of the running buffer is to allow nucleic acids to move through the agarose matrix. Therefore, the agarose gel must be submerged in the buffer.  The running buffer maintains the pH and ion concentration during electrophoresis. The pH stays within the appropriate range which is important because the changes in pH affect the net charge of the nucleic acids.  The running buffer contains EDTA (ethylenediaminetetraacetic acid) prevents nuclease from degrading the nucleic acids. TAE works better for cloning, because TBE contains borate.  The main running buffers are: TAE and TBE: Borate in TBE is an inhibitor for many enzymes, such as ligase. ▪ TAE is based on acetic acid. TAE produces a better separation of TAE works better for performing DNA ▪ TBE is based on boric acid. larger fragments (>3kb). extraction from agarose gel. TBE is suitable for obtaining a higher TBE has a higher buffering capacity - resolution of smaller fragments (