Nucleic Acid Techniques 1 Polymerase Chain Reaction PCR 1 What Is the Polymerase Chain Reaction PCR What if you don t have enough DNA for cloning purposes or Southern hybridization PCR The small sample of DNA can serve as polymerase template for DNA polymerase Make complementary primers add DNA Add primers in more than 1000 fold excess Heat to separate dsDNA stran ds then cool Run DNA polymerase usually Taq reaction again Repeat heating cooling polymerase cycle What Is the Polymerase Chain Reaction PCR Polymerase chain reaction PCR Polymerase Chain Reaction PCR Procedure for amplifying a segment of DNA by repeated rounds of replication Reaction Mixture DNA Two flanking primers Heat stable DNA polymerase The four dNTPs 1 Cycle has 3 temp dependent steps 1 Denaturation separation of 2 Annealing primers anneal to DNA strands DNA strands 3 Extension DNA polymerase synthesizes complementary DNA strands 4 Polymerase Chain Reaction PCR 20 cycles of PCR will increase the amount of target DNA template DNA around a million fold 220 Uses Cloning Diagnose infections diseases Detect rare pathological events such as mutations leading to cancer Forensics 5 Template Strand 5 5 3 Primer Design 3 5 5 5 3 ATTAGC CGAC TGGGTACGGG ATGCCC 5 3 5 ATTAGC 5 TAATCGGCTG ACCCATGCCC Two Primers described with respect to the template strand 1 Forward primer matches the sequence of 5 3 template strand at the start of the DNA sequence is complementary and therefore binds the 3 5 strand 2 Reverse primer matches the sequence of 3 5 strand at the end of the DNA sequence is complementary and therefore binds the 5 3 strand Template Strand 5 ATTAGCCGAC TGGGTACGGG 3 Forward Primer 5 ATTAGC 3 Reverse Primer 5 CCCGTA 3 Always right primers sequence in the 5 3 direction Convention 6 Electrophoretic analysis of DNA DNA is a polyanion charge so fragments migrates through an gel like matrix towards the anode charge in response to an electric field fragments separate based on size Gel like matrix 1 Agarose 2 Polyacrylamide Fragments with less kb migrate faster 7 Intercalating Agents Distort the Double Helix The structures of ethidium bromide acridine orange and actinomycin D three intercalating agents and their effects on DNA structure Ethidium Bromide used to aid in visualization of DNA separated using gel electrophoresis Acridine Orange is used in staining nucleic acids helpful in determining cell cycle Actinomycin D is an antibiotic shown to have anti cancer affects It is shown to have the ability to inhibit transcription by binding DNA at the transcription initiation complex and preventing elongation 8 2 Recombinant DNA Technology 9 Restriction Enzymes Bacteria have learned to restrict the possibility of attack from foreign DNA by means of restriction enzymes Type II and III restriction enzymes cleave DNA chains at selected sites Enzymes may recognize 4 6 or more bases in selecting sites for cleavage An enzyme that recognizes a 6 base sequence is a six cutter Nucleases Enzymes that hydrolyze nucleic acids are called Nucleases Endo cleave at an internal location Exo cleave at the end DNase act only on DNA RNase act only on RNA 11 Restriction Enzymes Restriction endonucleases Cleave double stranded DNA at an internal location Isolated chiefly from bacteria Three types Type I requires ATP non specific cleavage Type II no ATP requirement specific cleavage Type III requires ATP specific cleavage 12 Type II Restriction Endonucleases Enzymes may recognize 4 6 or more bases in selecting sites for cleavage No ATP requirement sticky ends have overhangs Cleavage can leave staggered or sticky ends or can produce blunt ends blunt ends have no overhangs 13 You do not need to memorize recognition sequences but 14 rather how to read them In Vitro Mutagenesis One method of PCR based site directed mutagenesis 1 Template DNA strands are separated and amplified by PCR 2 Following many cycles of PCR the DNA product can be used to transform E coli cells 3 The plasmid DNA can be isolated and screened for the presence of the unique restriction site by restriction endonuclease cleavage 12 1 What Does It Mean To Clone Clone a collection of molecules or cells all identical to an original molecule or cell To clone a gene is to make many copies of it for example in a population of bacteria Gene can be an exact copy of a natural gene Gene can be an altered version of a natural gene possible Recombinant DNA technology makes it Plasmids Are Very Useful in Cloning Genes Plasmids are naturally occurring extrachromosomal DNA Plasmids are circular dsDNA Plasmids can be cleaved by restriction enzymes leaving sticky Artificial plasmids can be constructed by linking new DNA These recombinant molecules can be autonomously replicated ends fragments to the sticky ends of plasmid and hence propagated Cloning Vectors Cloning vectors are plasmids that can be modified to carry new genes Plasmids useful as cloning vectors must have a replicator origin of a selectable marker replication antibiotic resistance gene A cloning site site where insertion of foreign DNA will not disrupt replication or inactivate essential markers Recombinant DNA Technology Definition The isolation amplification and modificiation of specific DNA sequences Also referred to molecular cloning or genetic engineering Construction of a recombinant DNA molecule 19 Recombinant DNA The 5 Basics of Cloning this is know as the vector 1 Must be able to cut and rejoin DNA a precise locations Must use sequence specific endonucleases called restriction endonucleases on foreign DNA and vector 2 Select a DNA molecule to serve as a carrier 3 Prepare and insert foreign DNA Use DNA ligase to join two pieces of DNA vector with inserted foreign DNA is known as recombinant DNA Introduce vector insert into host organism this process is known as transformation 4 5 Screen for host cells replicating the hybrid DNA 20 Recombinant DNA Choose an appropriate vector and clone foreign DNA 1 Plasmid 2 Bacteriophage 3 Bacterial artificial chromosomes BACs 21 1 Plasmid Clone DNA segments up to 10 kb Res Endonucleas e must produce overhangs sticky ends Relatively small Replicate easily Carry resistance gene s for antibiotics Contains multiple restriction sites 22 Biologically Functional Chimeric Plasmids Plasmids can be used to transform recipient E coli cells Transformation means the uptake and replication of exogenous DNA by a recipient cell To facilitate transformation the bacterial cells are rendered somewhat permeable to