Molecular Techniques Reminder: All molecular techniques are based on the chemical “personality” (or chemical properties) of the DNA molecule (or nucleic acids)

Organelle level Cellular level Studies of cell Cell fractionation Purification/ Identification Structure/ Function Organelle level Cell fractionation Nucleus Mitochondria Ceell membrane Cytosol Cellular level Microscope Molecular level: Macromolecules Proteins Carbohydrates Lipids Nucleic acids Atomic level C, H, O, N, S, P

- - - - Nucleic Acids Various lengths Negatively-charged phosphate-sugar backbone Various lengths - - - - Specificity of nucleotides Hydrogen bonds Nucleic Acids

CONTENTS Enzymes Electrophoresis Blotting and Hybridization Polymerase Chain Reaction DNA Sequences

Enzymes Large molecules made of various amino acids Act as catalysts to speed up reactions w/out being destroyed Increase the rate of reaction Highly specific Lowers energy of activation level Activity lost if denatured May contain cofactors such as metal ions or organic (vitamins)

Name of Enzymes End in –ase Identifies a reacting substance 1. sucrase – reacts sucrose 2. lipase - reacts lipid Describes function of enzyme 1. oxidase – catalyzes oxidation 2. hydrolase – catalyzes hydrolysis

Classification of Enzymes Class Reactions catalyzed Oxidoreductoases oxidation-reduction Transferases transfer group of atoms Hydrolases hydrolysis Lyases add/remove atoms to/from a double bond Isomerases rearrange atoms Ligases combine molecules

Enzyme Action: Lock and Key Model An enzyme binds a substrate in a region called the active site Only certain substrates can fit the active site Amino acid R groups in the active site help substrate bind Enzyme-substrate complex forms Substrate reacts to form product Product is released

Lock and Key Model + + E + S ES complex E + P P S S P

Enzymes use in Molecular Genetics 1. Restriction endonucleases/enzymes 2. Ligase 3. DNA polymerase

Restriction Enzymes Molecular scissors which isolated from bacteria where they are used as Bacterial defense against viruses Molecular scalpels to cut DNA in a precise and predictable manner Enzyme produced by bacteria that typically recognize specific 4-8 base pair sequences called restriction sites, and then cleave both DNA strands at this site A class of endo-nucleases that cleavage DNA after recognizing a specific sequence Members of the class of nucleases

Nuclease Breaking the phosphodiester bonds that link adjacent nucleotides in DNA and RNA molecules Endonuclease Cleave nucleic acids at internal position Exonuclease Progressively digest from the ends of the nucleic acid molecules

Endonuclease Type Characteristics I Have both restriction and modification activity Cut at sites 1000 nucleotides or more away from recognition site ATP is required II It has only restriction site activity Its cut is predictable and consistent manner at a site within or adjacent to restriction site It require only magnesium ion as cofactor III Cut at sites closed to recognition site

Restriction Enzymes There are already more than 1200 type II enzymes isolated from prokaryotic organism They recognize more than 130 different nucleotide sequence They scan a DNA molecule, stopping only when it recognizes a specific sequence of nucleotides that are composed of symetrical, palindromic sequence Palindromic sequence: The sequence read forward on one DNA strand is identical to the sequence read in the opposite direction on the complementary strand To Avoid confusion, restriction endo-nucleases are named according to the following nomenclature

Nomenclature The first letter is the initial letter of the genus name of the organism from which the enzyme is isolated The second and third letters are usually the initial letters of the organisms species name. It is written in italic A fourth letter, if any, indicates a particular strain organism Originally, roman numerals were meant to indicate the order in which enzymes, isolated from the same organisms and strain, are eluted from a chromatography column. More often, the roman numerals indicate the order of discovery

Nomenclature EcoRI E : Genus Escherichia co: Species coli R : Strain RY13 I : First endonuclease isolated BamHI B : Genus Bacillus am: species amyloliquefaciens H : Strain H I : First endonuclease isolated HindIII H : Genus Haemophilus in : species influenzae d : strain Rd III : Third endonuclease isolated

Specificity Enzyme Source Sequence End BamHI Bacillus amyloliquefaciens H GGATCC Sticky BglII Bacillus globigii AGATCT EcoRI Escherichia coli RY13 GAATTC EcoRII Escherichia coli R245 CCTGG HaeIII Haemophilus aegyptius GGCC Blunt HindII Haemophilus influenzae Rd GTPyPuAC HindIII AAGCTT HpaII Haemophilus parainfluenzae CCGG NotI Nocardia otitidis-caviarum GCGGCCGC PstI Providencia stuartii 164 CTGCAG

Restriction Product

Restriction enzymes can be grouped by: number of nucleotides recognized (4, 6,8 base-cutters most common) kind of ends produced (5’ or 3’ overhang (cohesive=sticky), blunt=flush) degenerate or specific sequences whether cleavage occurs within the recognition sequence

A restriction enzyme (EcoRI) 1. 6-base cutter 2. Specific palindromic sequence (5’GAATTC) 3. Cuts within the recognition sequence (type II enzyme) 4. produces a 5’ overhang (sticky end)

Ligase Any of a class of enzymes that act as catalysts in chemical reactions in which molecules are linked together, as in the synthesis and repair of DNA or in the formation of recombinant DNA Any of a class of enzymes that catalyze the linkage of two molecules, generally utilizing ATP as the energy donor (synthetase).

Function of DNA ligase The enzyme, DNA ligase, repairs the millions of DNA breaks generated during the normal course of a cell's life, for example, linking together the abundant DNA fragments formed during replication of the genetic material in dividing cells.

Ligase EC 6 Ligases EC 6.1 Forming carbon—oxygen bonds EC 6.2 Forming carbon—sulfur bonds EC 6.3 Forming carbon—nitrogen bonds EC 6.4 Forming carbon—carbon bonds EC 6.5 Forming phosphoric ester bonds EC 6.6 Forming nitrogen—metal bonds

DNA Ligase Mechanism

DNA Ligase Mechanism

Human DNA Ligase Human DNA ligase III gene encodes both nuclear and mitochondrial enzymes. DNA ligase plays a central role in DNA replication, recombination, and DNA repair.

DNA Polymerase an enzyme that is template based and has both 5’->3' DNA polymerase activity and 3’->5' exonuclease activity. highly processive, meaning it synthesizes long stretches of DNA without dissociating from the DNA template. an open right hand, composed of a thumb domain that binds to thioredoxin, a finger domain in which catalytic activity resides, a palm domain that cradles the DNA, and a terminal exonuclease domain 

Three main features of the DNA synthesis reaction DNA polymerase I catalyzes formation of phosphodiester bond between 3’-OH of the deoxyribose (on the last nucleotide) and he 5’-phosphate of the dNTP. Energy for this reaction is derived from the release of two of the three phosphates of the dNTP. DNA polymerase “finds” the correct complementary dNTP at each step in the lengthening process. rate ≤ 800 dNTPs/second low error rate 3. Direction of synthesis is 5’ to 3’

DNA elongation

DNA elongation

Types of DNA polymerase Polymerase Polymerization Exonuclease Exonuclease #Copies (5’-3’) (3’-5’) (5’-3’) I Yes Yes Yes 400 II Yes Yes No ? III Yes Yes No 10-20 3’ to 5’ exonuclease activity : ability to remove nucleotides from the 3’ end of the chain Important proofreading ability Without proofreading error rate (mutation rate) is 1 x 10-6 With proofreading error rate is 1 x 10-9 (1000-fold decrease) 5’ to 3’ exonuclease activity : the ability in DNA replication & repair.

Eukaryotic enzymes Five common DNA polymerases from mammals. Polymerase  (alpha): nuclear, DNA replication, no proofreading Polymerase  (beta): nuclear, DNA repair, no proofreading Polymerase  (gamma): mitochondria, DNA repl., proofreading Polymerase  (delta): nuclear, DNA replication, proofreading Polymerase  (epsilon): nuclear, DNA repair (?), proofreading Different polymerases for the nucleus and mtDNA Some polymerases proofread; others do not. Some polymerases used for replication; others for repair. Polymerases vary by species.

In this illustration, DNA ligase (in color) encircles the DNA double helix. Researchers investigating an important DNA-repair enzyme now have a better picture of the final steps of a process that glues together, or ligates, the ends of DNA strands to restore the double helix. The enzyme, DNA ligase, repairs the millions of DNA breaks generated during the normal course of a cell's life, for example, linking together the abundant DNA fragments formed during replication of the genetic material in dividing cells.

GEL ELECTROPHORESIS The motion of disperse charged particle relatives to a fluid under the influence of a spatially uniform electric field First observed by Reuss, 1807 For separating disperse charged biological molecule of any size/length Uses electricity Uses a matrix Uses buffer solution

- + Electrophoresis Factors affecting the mobility of molecules: 1. Molecular factors Charge Size Shape 2. Environment factors Electric field strength Matrix (pore: sieving effect) Running buffer +

Electrophoresis

Types of matrix (supporting media) Paper Agarose 1. purified large MW polysaccharide (from agar) 2. very open (large pore) gel 3. used frequently for large DNA molecules Acrylamide 1. a white odorless crystalline solid chemical compound 2. soluble in water, ethanol, ether, chloroform 3. used to synthesize poly-acrylamide which find many uses as water soluble thickeners Starch Cellulose acetate

An analytical technique used to separate DNA by size DNA Agarose Gel An analytical technique used to separate DNA by size Electric field induces DNA to migrate toward the anode due to the net negative charge of the sugar phosphate backbone of the DNA Longer molecules migrate more slowly Visualized using a fluorescence dye special for DNA such as ethidium bromide

Polyacrylamide Gels acrylamide polymer very stable gel can be made at a wide variety of concentrations large variety of pore sizes (powerful sieving effect)

SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Sodium Dodecyl Sulfate = Sodium Lauryl Sulfate: CH3(CH2)11SO3- Na+ Amphipathic molecule Strong detergent to denature proteins Binding ratio: 1.4 g SDS/g protein Charge and shape normalization

Isoelectric Focusing Electrophoresis (IFE) Separate molecules according to their isoelectric point (pI) At isoelectric point (pI) molecule has no charge (q=0), hence molecule ceases pH gradient medium

2-dimensional Gel Electrophoresis First dimension is IFE (separated by charge) Second dimension is SDS-PAGE (separated by size) So called 2D-PAGE High throughput electrophoresis, high resolution

2-dimensional Gel Electrophoresis Spot coordination pH MW

Blotting and Hybridization

Transfer the DNA from the gel to a solid support Blotting Transfer the DNA from the gel to a solid support Transferring of DNA, RNA, Protein to an immobilizing binding matrix such as nitrocellulose paper or nylon Northern blot (RNA) Western blot (Protein) Eastern blot (???) Southern blot (DNA)

Blotting Two methods : Capillary transfer Electrophoretic transfer

SOUTHERN BLOTTING The technique was developed by E.M. Southern in 1975. The Southern blot is used to detect the presence of a particular piece of DNA in a sample. The DNA detected can be a single gene, or it can be part of a larger piece of DNA such as a viral genome The key to this method is hybridization. Hybridization-process of forming a double-stranded DNA molecule between a single-stranded DNA probe and a single-stranded target patient DNA.

There are 2 important features of hybridization: SOUTHERN BLOTTING There are 2 important features of hybridization: The reactions are specific The probes will only bind to targets with a complementary sequence. The probe can find one molecule of target in a mixture of millions of related but non-complementary molecules.

Southerns Blotting (DNA Blotting) DNA fragments created by restriction digestion are separated on an agarose gel Separated fragments are denatured and transferred to a membrane (blot) by blotting Probe is hybridized to complementary sequences on the blot and excess probe is washed away Location of probe is determined by detection method (e.g., film, fluorometer)

Southern blotting 20

Some Applications of DNA Blots Map restrictions sites near a particular locus for gene isolation or allele analysis (e.g., RFLP restriction fragment length polymorphism) Identity of closely related genes Confirmation of gene transfer or gene disruption Detection of foreign DNA

RNA Blotting (Northern) Use DNA to prove RNA RNA is separated by size on a denaturing agarose gel and then transferred onto a membrane (blot) Probe is hybridized to complementary sequences on the blot and excess probe is washed away Location of probe is determined by detection method (e.g., film, fluorometer)

RNA Blotting (Northern) RNA Mixture RNA

RNA Blotting (Northern) Advantage: - Very sensitive - Blots are reusable - Technical protocol is relatively simple - Can detect mRNA splice variants Disadvantage: Use of radioactivity (although non-radioactive techniques are available) - Laborious if many genes need to be tested - Assay is time-consuming

Applications of RNA Blots Detect the expression level and transcript size of a specific gene in a specific tissue or at a specific time. Sometimes mutations do not affect coding regions but transcriptional regulatory sequences (e.g., UAS/URS, promoter, splice sites, copy number, transcript stability)

Western Blot Protein blotting Highly specific qualitative test Can determine if above or below threshold Typically used for research Use denaturing SDS-PAGE Solubilizes, removes aggregates & adventitious proteins are eliminated Components of the gel are then transferred to a solid support or transfer membrane weight Paper towel Wet filter paper Transfer membrane Paper towel

Western Blot

Pairing of complementary DNA and/or RNA and/or protein Hybridization Pairing of complementary DNA and/or RNA and/or protein

Hybridization It can be DNA:DNA, DNA:RNA, or RNA:RNA (RNA is easily degraded) It depended on the extent of complementation It depended on temperature, salt concentration, and solvents Small changes in the above factors can be used to discriminate between different sequences (e.g. small mutations can be detected) Probes can be labeled with radioactivity, fluorescent dyes, enzymes. Probes can be isolated or synthesized sequences

In-situ hybridisation Hybridization which is performed by denaturing the DNA of cell squash on a microscope slide so that reaction is possible with an added of probe Chromosome in-situ hybridisation DNA probe detects sequences in chromosomes Map gene sequences Tissue in-situ hybridisation RNA probe detects sequences in cells and tissues Identify sites of gene expression Analyse tissue distribution of expression 22

Oligonucleotide probes Single stranded DNA (usually 15-40 bp) Degenerate oligo-nucleotide probes can be used to identify genes encoding characterized proteins Use amino acid sequence to predict possible DNA sequences Hybridize with a combination of probes TT(T/C) - TGG - ATG - GA(T/C) - TG(T/C) - could be used for FWMDC amino acid sequence Can specifically detect single nucleotide changes

Detection of Probes Probes can be labeled with radioactivity, fluorescent dyes, enzymes. Radioactivity is often detected by X-ray film (autoradiography) Fluorescent dyes can be detected by fluorometers, scanners Enzymatic activities are often detected by the production of dyes or light (x-ray film)

Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction Powerful technique for amplifying DNA Amplified DNA are then separated by gel electrophoresis

Each cycle the amount of DNA doubles PCR A simple rapid, sensitive and versatile in vitro method for selectively amplifying defined sequences/regions of DNA/RNA from an initial complex source of nucleic acid - generates sufficient for subsequent analysis and/or manipulation Amplification of a small amount of DNA using specific DNA primers (a common method of creating copies of specific fragments of DNA) DNA fragments are synthesized in vitro by repeated reactions of DNA synthesis (It rapidly amplifies a single DNA molecule into many billions of molecules) In one application of the technology, small samples of DNA, such as those found in a strand of hair at a crime scene, can produce sufficient copies to carry out forensic tests. Each cycle the amount of DNA doubles

Background on PCR The ability to generate identical high copy number DNAs made possible in the 1970s by recombinant DNA technology (i.e., cloning). Cloning DNA is time consuming and expensive Probing libraries can be like hunting for a needle in a haystack. Requires only simple, inexpensive ingredients and a couple hours PCR, “discovered” in 1983 by Kary Mullis, Nobel Prize for Chemistry (1993). It can be performed by hand or in a machine called a thermal cycler.

Three Steps Separation Priming Copying Double Stranded DNA is denatured by heat into single strands. Short Primers for DNA replication are added to the mixture. Priming DNA polymerase catalyzes the production of complementary new strands. Copying The process is repeated for each new strand created All three steps are carried out in the same vial but at different temperatures

Magnesium as a Cofactor Step 1: Separation Combine Target Sequence, DNA primers template, dNTPs, Taq Polymerase Target Sequence 1. Usually fewer than 3000 bp 2. Identified by a specific pair of DNA primers- usually oligonucleotides that are about 20 nucleotides Heat to 95°C to separate strands (for 0.5-2 minutes) Longer times increase denaturation but decrease enzyme and template Magnesium as a Cofactor Mg stabilizes the reaction between: oligonucleotides and template DNA DNA Polymerase and template DNA

Denatures DNA by uncoiling the Double Helix strands. Heat Denatures DNA by uncoiling the Double Helix strands.

Step 2: Priming Decrease temperature by 15-25 °C Primers anneal to the end of the strand 0.5-2 minutes Shorter time increases specificity but decreases yield Requires knowledge of the base sequences of the 3’ - end

Selecting a Primer Primer length Melting Temperature (Tm) Specificity Complementary Primer Sequences G/C content and Polypyrimidine (T, C) or polypurine (A, G) stretches 3’-end Sequence Single-stranded DNA

Step 3: Polymerization Since the Taq polymerase works best at around 75 ° C (the temperature of the hot springs where the bacterium was discovered), the temperature of the vial is raised to 72-75 °C The DNA polymerase recognizes the primer and makes a complementary copy of the template which is now single stranded. Approximately 150 nucleotides/sec

Potential Problems with Taq Lack of proof-reading of newly synthesized DNA. Potentially can include di-Nucleotriphosphates (dNTPs) that are not complementary to the original strand. Errors in coding result Recently discovered thermostable DNA polymerases, Tth and Pfu, are less efficient, yet highly accurate.

How PCR works Begins with DNA containing a sequence to be amplified and a pair of synthetic oligonucleotide primers that flank the sequence. Next, denature the DNA at 94˚C. Rapidly cool the DNA (37-65˚C) and anneal primers to complementary s.s. sequences flanking the target DNA. Extend primers at 70-75˚C using a heat-resistant DNA polymerase (e.g., Taq polymerase derived from Thermus aquaticus). Repeat the cycle of denaturing, annealing, and extension 20-45 times to produce 1 million (220) to 35 trillion copies (245) of the target DNA. Extend the primers at 70-75˚C once more to allow incomplete extension products in the reaction mixture to extend completely. Cool to 4˚C and store or use amplified PCR product for analysis.

Thermal cycler protocol Example Step 1 7 min at 94˚C Initial Denature Step 2 45 cycles of: 20 sec at 94˚C Denature 20 sec at 64˚C Anneal 1 min at 72˚C Extension Step 3 7 min at 72˚C Final Extension Step 4 Infinite hold at 4˚C Storage

The Polymerase Chain Reaction

PCR amplification Each cycle the oligo-nucleotide primers bind most all templates due to the high primer concentration The generation of mg quantities of DNA can be achieved in ~30 cycles (~ 4 hrs)

THE REACTION COMPONENTS OPTIMISING PCR THE REACTION COMPONENTS Starting nucleic acid - DNA/RNA Thermo-stable DNA polymerase e.g. Taq polymerase Oligonucleotides (primer) Design them well! Buffer Tris-HCl (pH 7.6-8.0) Mg2+ dNTPs (dATP, dCTP, dGTP, dTTP)

RAW MATERIAL Organims, Organ, Tissue, cells (hair root, callus, leaves, root, seed) Obtain the best starting material. Some can contain inhibitors of PCR, so they must be removed e.g. Haem in blood Good quality genomic DNA if possible Empirically determine the amount to add

POLYMERASE Number of options available Taq polymerase Pfu polymerase Tth polymerase How big is the product? 100bp 40-50kb What is end purpose of PCR? 1. Sequencing - mutation detection -. Need high fidelity polymerase -. integral 3’ ________ 5' proofreading exonuclease activity 2. Cloning 3. Marker development

Length ~ 10-30 nucleotides (21 nucleotides for gene isolation) PRIMER DESIGN Length ~ 10-30 nucleotides (21 nucleotides for gene isolation) Base composition: 50 - 60% GC rich, pairs should have equivalent Tms Tm = [(number of A+T residues) x 2 °C] + [(number of G+C residues) x 4 °C] Initial use Tm–5°C Avoid internal hairpin structures No secondary structure Avoid a T at the 3’ end Avoid overlapping 3’ ends – will form primer dimers Can modify 5’ ends to add restriction sites

PRIMER DESIGNER Sci. Ed software Use specific programs OLIGO Medprobe PRIMER DESIGNER Sci. Ed software Also available on the internet http://www.hgmp.mrc.ac.uk/GenomeWeb/nuc-primer.html

Mg2+ CONCENTRATION Normally, 1.5mM MgCl2 is optimal Best supplied as separate tube Always vortex thawed MgCl2 Mg2+ concentration will be affected by the amount of DNA, primers and nucleotides

USE MASTERMIXES WHERE POSSIBLE

PCR can amplify a single DNA molecule, e.g. from a single sperm. How Powerful is PCR? PCR can amplify a usable amount of DNA (visible by gel electrophoresis) in ~2 hours. The template DNA need not be highly purified — a boiled bacterial colony. The PCR product can be digested with restriction enzymes, sequenced or cloned. PCR can amplify a single DNA molecule, e.g. from a single sperm.

Applications of PCR Amplify specific DNA sequences (genomic DNA, cDNA, recombinant DNA, etc.) for analysis 1. Gene isolation 2. Fingerprint development Introduce sequence changes at the ends of fragments Rapidly detect differences in DNA sequences (e.g., length) for identifying diseases or individuals Identify and isolate genes using degenerate oligonucleotide primers Design mixture of primers to bind DNA encoding conserved protein motifs Genetic diagnosis - Mutation detection The basis for many techniques to detect gene mutations (sequencing) - 1/6 X 10-9 bp

Applications of PCR Paternity testing Mutagenesis to investigate protein function Quantify differences in gene expression → Reverse transcription (RT)-PCR Identify changes in expression of unknown genes → Differential display (DD)-PCR  Forensic analysis at scene of crime Industrial quality control DNA sequencing

DNA Sequencing

DNA sequencing Determination of nucleotide sequence the determination of the precise sequence of nucleotides in a sample of DNA Two similar methods: 1. Maxam and Gilbert method 2. Sanger method They depend on the production of a mixture of oligonucleotides labeled either radioactively or fluorescein, with one common end and differing in length by a single nucleotide at the other end This mixture of oligonucleotides is separated by high resolution electrophoresis on polyacrilamide gels and the position of the bands determined 11

The Maxam-Gilbert Technique Principle: Chemical Degradation of Purines Purines (A, G) damaged by dimethylsulfate Methylation of base Heat releases base Alkali cleaves G Dilute acid cleave A>G

Maxam-Gilbert Technique Pyrimidines (C, T) are damaged by hydrazine Piperidine cleaves the backbone 2 M NaCl inhibits the reaction with T

Maxam and Gilbert Method Chemical degradation of purified fragments (chemical degradation) The single stranded DNA fragment to be sequenced is end-labeled by treatment with alkaline phosphatase to remove the 5’phosphate It is then followed by reaction with P-labeled ATP in the presence of polynucleotide kinase, which attaches P labeled to the 5’terminal The labeled DNA fragment is then divided into four aliquots, each of which is treated with a reagent which modifies a specific base 1. Aliquot A + dimethyl sulphate, which methylates guanine residue 2. Aliquot B + formic acid, which modifies adenine and guanine residues 3. Aliquot C + Hydrazine, which modifies thymine + cytosine residues 4. Aliquot D + Hydrazine + 5 mol/l NaCl, which makes the reaction specific for cytosine The four are incubated with piperidine which cleaves the sugar phosphate backbone of DNA next to the residue that has been modified 11

Maxam-Gilbert sequencing - modifications

Maxam-Gilbert sequencing: Summary

Advantages/disadvantages Maxam-Gilbert sequencing Requires lots of purified DNA, and many intermediate purification steps Relatively short readings Automation not available (sequencers) Remaining use for ‘footprinting’ (partial protection against DNA modification when proteins bind to specific regions, and that produce ‘holes’ in the sequence ladder) In contrast, the Sanger sequencing methodology requires little if any DNA purification, no restriction digests, and no labeling of the DNA sequencing template

Sanger Fred Sanger, 1958 1980 dideoxy sequencing Was originally a protein chemist Made his first mark in sequencing proteins Made his second mark in sequencing RNA 1980 dideoxy sequencing

Original Sanger Method Random incorporation of a dideoxynucleoside triphosphate into a growing strand of DNA Requires DNA polymerase I Requires a cloning vector with initial primer (M13, high yield bacteriophage, modified by adding: beta-galactosidase screening, polylinker) Uses 32P-deoxynucleoside triphosphates

Sanger Method in-vitro DNA synthesis using ‘terminators’, use of dideoxi- nucleotides that do not permit chain elongation after their integration DNA synthesis using deoxy- and dideoxynucleotides that results in termination of synthesis at specific nucleotides Requires a primer, DNA polymerase, a template, a mixture of nucleotides, and detection system Incorporation of di-deoxynucleotides into growing strand terminates synthesis Synthesized strand sizes are determined for each di-deoxynucleotide by using gel or capillary electrophoresis Enzymatic methods

Dideoxynucleotide 5’ BASE PPP O CH2 O 3’ no hydroxyl group at 3’ end prevents strand extension 12

The principles Partial copies of DNA fragments made with DNA polymerase Collection of DNA fragments that terminate with A,C,G or T using ddNTP Separate by gel electrophoresis Read DNA sequence 13

3’ CCGTAC 5’ primer 5’ 3’ dNTP ddTTP ddCTP ddGTP ddATP GGCA GGCAT GGC A T C G 14

Chain Terminator Basics Template-Primer Target TGCA ddA ddG ddC ddT Labeled Terminators Extend ddA A ddC dN : ddN 100 : 1 Ladder n, n+1... AC ddG ACG ddT

Electrophoresis

Sanger Method Sequencing Gel

Sequencing of DNA by the Sanger method

Comparison Sanger Method Maxam Gilbert Method Enzymatic Requires DNA synthesis Termination of chain elongation Maxam Gilbert Method Chemical Requires DNA Requires long stretches of DNA Breaks DNA at different nucleotides