Outline of last lecture DNA replication Conservative, semiconservative, dispersive replication Semiconservative – Meselson and Stahl, 1958 Analysis of replication by CsCl density gradient centrifugation Initiation and termination of replication 5’ to 3’ new strand synthesis Replication fork and Okazaki fragments grow

Polymerase Chain Reaction (PCR) Syed Abdullah Gilani

Objectives and Output What is PCR ? Steps before PCR, DNA extraction How PCR works? What are the reagents required for PCR? Steps after PCR, principles and performance of electrophoresis Applications of PCR

Steps before PCR DNA extraction nucleic acid two complementary nucleotide building block chains The nucleotides phosphate group, a five carbon sugar, and a nitrogen base

DNA Primary strand CCGAATGGGATGC GGCTTACCCTACG Complementary strand

DNA Molecule Adenine Thymine Guanine Cytosine

PCR PCR is a technique Takes specific sequence of DNA of small amounts and amplifies it to be used for further testing

PCR Targets targets in PCR sequences of DNA on each end of the region of interest, can be a complete gene or small sequence

TTAAGGCTCGA . . . . AATTGGTTAA PCR Targets The number of bases in the targets can vary. TTAAGGCTCGA . . . . AATTGGTTAA The . . . . Represents the middle DNA sequence, and does not have to be known to replicate it.

Steps of PCR 2. PCR Targets Denaturing Primers Annealing Cycles Requirements

PCR reagents mix

PCR Denaturing First step in PCR DNA strands separated by heating to 95°C

Denaturation

PCR Primers 15 – 30 nucleotides single-stranded Used for the complementary building blocks of the target sequence Forward and Reverse primers allows both strands to be copied simultaneously in both directions

PCR Primers TTAACGGCCTTAA . . . TTTAAACCGGTT AATTGCCGGAATT . . . . . . . . . .> and <. . . . . . . . . . AAATTTGGCCAA

PCR Annealing Annealing is the process of allowing two sequences of DNA to form hydrogen bonds. The annealing of the target sequences and primers is done by cooling the DNA to 55°C.

PCR Cycles

PCR Taq DNA Polymerase Thermus aquaticus 176 °F hot springs in Yellow Stone National Forest DNA polymerase amplifies the DNA from the primers by the polymerase chain reaction, in the presence of Mg

PCR Cycles

PCR Cycles

PCR cocktail Magnesium chloride 0.5-2.5 mM Buffer pH 8.3-8.8 dNTPs 20-200 µM Primers 0.1-0.5 µM DNA Polymerase 1-2.5 units Target DNA  1 µg

PCR Reaction Denaturing 94°- 95°C Primer Annealing 55°- 65°C Extension of DNA 72 °C Number of Cycles 25-40

Cycle 1 Cycle 2 Cycle 3 Denaturation Annealing Extension 5’ 3’ 5’ 3’

Number of DNA molecules Rate of PCR 2n Initial DNA 1 2 4 8 Number of DNA molecules

Copies of DNA=2N

Why do we run PCR? Why do we run 25-40 cycles why not a single cycle of amplification?

Resolution and Detection of Nucleic Acids

Gel Electrophoresis Electrophoresis is the movement of molecules by an electric current. Nucleic acid moves from a negative to a positive pole.

Gel Electrophoresis When DNA is applied to a macromolecular cage or gel such as agarose or polyacrylamide, its migration under the pull of the current is impeded.

The movement of molecules is impeded in the gel so that molecules will collect or form a band according to their speed of migration. % agarose: 2% 4% 5% 500 bp 200 bp 50 bp 500 bp 200 bp 50 bp 500 bp 200 bp 50 bp The concentration of gel/buffer will affect the resolution of fragments of different size ranges.

Gel Electrophoresis Slab gel electrophoresis can have either a horizontal or vertical format. Sample is introduced into wells at the top of the gel.

Polyacrylamide Gel Electrophoresis (PAGE) Acrylamide, in combination with a cross linker, methylene bis-acrylamide Synthetic, consistent polymer Polymerization catalysts: ammonium persulfate (APS) plus N,N,N',N'-tetramethylethylenediamine (TEMED), or light activation Resolves 1 bp difference in a 1 kb molecule (0.1% difference)

Electrophoresis Buffers Carry current and protect samples during electrophoresis. Tris Borate EDTA (TBE), Tris Acetate EDTA (TAE), Tris Phosphate EDTA (TPE) used most often for DNA. 10 mM sodium phosphate or MOPS buffer used for RNA. Buffer additives modify sample molecules Formamide, urea (denaturing agents)

Electrophoresis Equipment Horizontal or submarine gel

Electrophoresis Equipment Vertical gel

Electrophoresis Equipment Combs are used to put wells in the cast gel for sample loading. Regular comb: wells separated by an “ear” of gel Houndstooth comb: wells immediately adjacent

Running a Gel Use the proper gel concentration for sample size range. 0.5–5% agarose 3.5–20% polyacrylamide Use the proper comb (well) and gel size.

Running a Gel Load sample mixed with tracking dye (dye + density agent).

Running a Gel Detect bands by staining during or after electrophoresis Ethidium bromide: for double-stranded DNA SyBr green or SyBr gold: for single- or double-stranded DNA or for RNA Silver stain: more sensitive for single- or double-stranded DNA or for RNA and proteins

Summary Electrophoresis is used to separate molecules by size and/or charge. Nucleic acid fragments can be resolved on agarose of polyacrylamide gels. The choice of electrophoresis method depends on the type and size of sample.

Gel Electrophoresis Gel Electrophoresis

Applications of PCR Disease detection and identification (Neisseria gonorrhoeae, HIV-1, plant diseases etc.) Forensic testing Paternity Criminal identification Plant breeding Marker assisted selection – high yield, high disease resistant offspring QTL mapping Biodiversity and conservation Molecular systematics

Applications of PCR PCR can also be used in forensic testing. The DNA sequences used are of short repeating patterns called VNTR (variable number of tandem repeat), which can range from 4 to 40 nucleotides in different individuals.

Applications of PCR One set of VNTR locus are inherited from the mother and one set from the father. The genes are amplified using PCR, and then run through electrophoresis. The position of the two bands on the electrophoresis gel depends on the exact number of repeats at the locus.

Applications of PCR

Applications of PCR Three VNTR loci from suspects, along with the DNA from the scene are run through PCR amplification, and then through electrophoresis. This gives six bands, which can have common bands for some individuals, but the overall pattern is distinctive for each person.

Applications of PCR

Plant Molecular Systematics Types of molecular data? DNA sequences DNA restriction sites: RFLPs Allozymes - different forms of proteins Microsatellites - DNA regions w/tandem repeats RAPDs - Random Amplification of Polymorphic DNA AFLPs - Amplified Fragment Length Polymorphism

How are Plant Molecular Data Acquired? Plant collected: voucher prepared! Live samples, e.g., allozyme analysis Dried or liquid-preserved samples, e.g., DNA analysis

DNA Sequencing How does it work? Similar to PCR amplification But, small amount of Dideoxynucleotides used (along with higher conc. of nucleotides) Dideoxynucleotides, once joined to new DNA strand, terminate polymerase reaction. Dideoxynucleotides identified by fluorescence pattern. Length of DNA strands determined by electrophoresis.

DNA Sequencing

DNA Sequencing

Types of DNA sequence data Chloroplast cpDNA Nuclear nDNA Mitochondrial mtDNA (not used much with plants; used w/ animals)

Chloroplast DNA

Some Chloroplast genes: Gene Location Function atpB Large single-copy region of chloroplast Beta subunit of ATP synthethase, which functions in the synthesis of ATP via proton translocation rbcL Large single-copy region of chloroplast Large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (RUBISCO), which functions in the initial fixation of carbon dioxide in the dark reactions matK Large single-copy region of chloroplast Maturase, which functions in splicing type II introns from RNA transcripts ndhF Small single-copy region of chloroplast Subunit of chloroplast NADH dehydrogenase, which functions in converting NADH to NAD + H+, driving various reactions of respiration Today: Chloroplast inter-genic spacer regions used!

Nuclear DNA: ITS Sequence Data: used in lower-level analyses

DNA alignment