PCR Theory and Primer Design Lecture Notes PDF

Lecture 13 & 14: PCR Theory & Primer Design RDNA202 Cassie [email protected] Biotechnology and Genomics Lecture 12: DNA extraction & PCR Theory Lecture 13: PCR Theory Lecture 14: Primer Design Lecture 15: Tutorial / Lecture continuation PCR Variations – Types of PCR Reac...

Lecture 13 & 14: PCR Theory & Primer Design RDNA202 Cassie [email protected] Biotechnology and Genomics Lecture 12: DNA extraction & PCR Theory Lecture 13: PCR Theory Lecture 14: Primer Design Lecture 15: Tutorial / Lecture continuation PCR Variations – Types of PCR Reactions 1. Conventional (qualitative) PCR – Discussed in previous lectures 2. Multiplex PCR 3. Nested PCR 4. Inverse PCR 5. Reverse transcriptase PCR (RT-PCR) 6. Overlap PCR 7. Amplification Fragment Length Polymorphism (AFLP) 8. Many other types… PCR Variations – Types of PCR Reactions 2. Multiplex PCR Amplify multiple targets in a single PCR Allows simultaneous analysis 3. Nested PCR Modified PCR – intended to decrease non-specific binding Involves two PCR steps First reaction produces DNA product = template for second reaction Application examples: Amplify targeted sequences in small (microbial genome) DNA Amplify degraded DNA (Forensic samples) PCR Variations – Types of PCR Reactions 4. Inverse PCR Uses known sequences to identify unknown sequences E.g. determine retroviruses and transposons – that randomly integrate into genomic DNA Uses Restriction enzymes 5. Reverse transcriptase PCR (RT-PCR) Detect cell-specific gene expression From mRNA to cDNA mRNA = starting material – converted using reverse transcriptase into complementary DNA cDNA is then amplified via conventional PCR PCR Variations – Types of PCR Reactions 6. Overlap PCR/Overlap extension PCR Joining of more PCR produces together Involves splicing of DNA molecule Applications include: Cloning large complex fragments Making edits to cloned genes Fusing two gene elements together 7. Amplification Fragment Length Polymorphism (AFLP) Selective amplification of restriction fragments from restriction digested genomic DNA Polymorphism in different primers Highly sensitive – Detect polymorphisms - Used in microsatellite analyses  SOME uses of PCR Identification of a person based on few skin cells left behind after touching something. Evolutionary research Can amplify and examine DNA from mummies, mammoths, fossils etc. (Jurassic park?) Amplification of DNA from single embryonic cells Prenatal diagnosis Disease diagnosis e.g. HIV, Covid etc. Basic research into gene structure and genomes  Analysis of PCR Products – Gel electrophoresis Components of gel electrophoresis 1. PCR product (amplified DNA) 2. DNA ladder – Molecular marker 3. Agarose powder 4. Gel stain 5. Loading Dye 6. Gel buffer and electrophoresis buffer (TBE or TAE) 7. Electrophoresis set  Agarose Gel electrophoresis Analytical procedure Used in research, biomedical, and forensic laboratories Uses electric current (negative to positive) Separates DNA fragments based on size Used to determine size of DNA molecules Ranging from 100 to 30 000 bp  Agarose Gel electrophoresis – Making the gel Agarose powder dissolved with boiling buffer Buffer is either TBE or TAE Liquid mixture poured into a casting tray to solidify Wells made by inserting a comb into the liquid before it solidifies  Agarose Gel electrophoresis – Loading DNA & running DNA (PCR Product) Mixed with loading dye Loaded into wells of gel Sits between positive and negative electrodes Voltage and time are selected When electrical current applied DNA (negative) will move towards the positive electrode (anode)  DNA Molecular Sizes Small fragments – move faster through the gel Large fragments – move slower All different sizes spread out Single DNA molecule can have sequences with multiple genes – produce multiple fragments Migration of DNA molecule is inversely proportional to logarithm of lengths (bps) Calculating Log Molecular weight of Standards Used to Plot a Standard Curve Primer Design Oligonucleotide Primers – essential when running PCR Primers need to be complementary to template region/strand Primers must amplify unique sequence Unless you are doing fingerprinting – want lots of bands Chemically synthesised by joining nucleotides together  Primer Design Ta rg e t D N A (on ly o n e s tra n d re p res e n te d ) forw ard prim er sa m e se q u e n ce (se e b e lo w ) 5 ’..........AT...............G C..........[G G C A A....~ 3 0 0.....T C C A G ]..........G A.................C T......3 ’ ~ 10 nt reg io n to be a m plifi e d ~ 10 nt rev erse prim e r re ve rs e co m p le m e n t Take note on orientation of primers o f th is se q u e n ce (se e b e lo w ) S e q u e n c e o f fo rw a rd p rim e r - 5 ’AT...............G C S e q u e n c e o f re ve rs e p rim e r - 5 ’ A G...............T C Synthesis in 5’- 3’ direction re v e rs e p rim e r 3 ’C T................G A 5 ’ 5 ’..........AT...............G C..........[G G C A A.....~ 3 0 0.....T C C A G ].........G A................C T......3 ’ 3 ’..........TA...............C G..........[C C G T T......~ 3 00.....A G G T C ]..........C T.................G A......5 ’ 5 ’AT...............G C 3 ’ fo rw ard p rim er  Primer Design – Length Usually 18-30 nucleotides long 5’ A G C A C Determines specificity 3’ T C G T G Affects annealing to DNA template Too short – low specificity – leads to non-specific amplification Too long – Reduces template-binding efficiency at normal annealing temperature Increases probability of secondary structure formation e.g. hairpins – caused by intrastrand self-complementarity  Primer Design – Composition Primers must not bind each other = interstrand complementarity This prevents them from binding DNA Primers must have GC content between 40-60% (ideal = 50- 60%) This is the percentage of guanine or cytosine in DNA or RNA molecule GC content often used to predict5’ annealing A A G temperature C A C T C G T G G G 5’ GC content calculated as follows: GC% - (G+C)/length of sequence  Primer Design – Melting Temperature (Tm) Primers must have a Tm between 45-70°C This is the temperature where 50% of DNA duplex becomes single stranded Determined by: Primer length Base composition Concentration or primers Simple formula for Tm calculation: For shorter than 18 bases: Tm = 2(A+T) + 4(G+C) For longer primers: Tm = 64.9 + 41(G+C-16.4)/(A+T+G+C)  Primer Design – Annealing Temperature (Ta) Primers must have a Ta between 50-68°C Temperature where primers bind to complementary DNA regions of interest Depends directly on: Primer length & Base composition Ta = 5°C below lowest Tm of primer pair If Ta is: Too high – May cause insufficient primer-template hybridization = Low PCR yield Too low – May result in non-specific products Optimal annealing temperature for any primer pair on target calculated by: TaOpt = 0.3 x (Tm of primer) + 0.7 x (Tm of product) - 25 Primer Design – Properties Primers should be 18 – 30 bp in length GC content ideally should be 40-60% (G+C) Tms should be between 45-70°C Tm should be within 5°C of each other Primers should End (3’ end) in G or C or GC of CG Prevents “breathing” of ends & increases priming efficiency Primers should not have complementary regions Runs of 3 or more Cs or Gs at 3’ ends - avoided May promote mispriming at G or C-rich sequences Due to stability of annealing Primer Design Tools Online tools that assist in Primer creation based on a specific DNA sequence. E.g. Primer Blast Eurofins Genomics PCR Primer Design Tool Primer3web Different Types of Primers used in PCR 1. Universal Primer Binds a variety of DNA templates 2. Species-specific Primer Designed to amplify only one product Designed for a specific gene target in a specific species Different Types of Primers used in PCR 3. Oligo dT Primers Oligo d (T) 12-18 is classic primer Used to Prime synthesis of first cDNA strand by reverse Transcriptase Uses Poly A+ mRNA as template 4. Degenerate Primers Mixtures of similar – but not identical – primers Convenient if same gene is to be amplified from different organisms Gene likely to be similar but not identical Next Section: DNA Sequencing RDNA202 Cassie [email protected]