Lectures 22-24 BCM 475 - M001
Popular in Biochemistry I
Popular in Biochemistry
BCM 475 - M001
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This 14 page Class Notes was uploaded by Annie Notetaker on Saturday November 14, 2015. The Class Notes belongs to BCM 475 - M001 at Syracuse University taught by M. Braiman, R. Welch in Fall 2015. Since its upload, it has received 59 views. For similar materials see Biochemistry I in Biochemistry at Syracuse University.
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Date Created: 11/14/15
Lecture 10/26: Genetic Information II (p. 121-134) DNA and RNA Tertiary Structure • Closed circular DNA -E.g. plasmids • Supercoiled DNA -Closed circular DNA -Compact DNA -“Supercoils must be relaxed when DNA is replicated—requires enzymes” • Relaxed DNA -Closed circular DNA -“Extremely long DNA (e.g. eukaryotic chromosomes) takes so long to relax twisting on its own, that it also requires enzymes” Structures of Single-Stranded Nucleic Acids • E.g. stem-loop (hairpin loop) structure “created when two complementary sequences within a single strand (of DNA or RNA) come together to form double-helical structures” • Figure 4.18 Complex Structures of RNA Molecules • RNA molecules commonly adopt folded conformations • E.g. hairpins, bulges, internal loops, pseudoknots, multibranched loops, coaxial stacking of stems • Pseudoknots, as their name suggests, are “not topologically entangled”; a pull at the 5’ and 3’ end will straighten the knot out The Double Helix and the Transfer of Genetic Information • Because a particular base on one strand of DNA is always paired with a specific base on an adjacent strand, “the sequence of bases of one strand of the double helix precisely determines the sequence of the other strand” • DNA is replicated via semiconservative replication -In every generation, one parent strand is conserved in the daughter strands DNA Replication/ Phosphodiester-Bridge Formation • Catalyzed by DNA polymerases • Reaction: (DNA) +ndNTP (any deoxyribonucleotide) ⇌ (DNA) n+1+ PP ipyrophosphate ion) • Characteristics of DNA synthesis: 1. The 2+oxynucleoside 5’-triphosphates dATP, dGTP, dCTP, and TTP (as well as Mg ) are required 2. The newly synthesized DNA strand is complementary to a preexisting template strand -DNA polymerase à template-directed enzyme 3. “A primer strand having a free 3’-OH group must be already bound to the template strand” 1 4. “Many DNA polymerases are able to correct mistakes in DNA by removing mismatched nucleotides” 5. Direction of synthesis: 5’ à 3’ • Chain-elongation reaction (DNA) +ndNTP ⇌ (DNA) n+1 + PP i Q = [(DNA) n+1][ PPi/ [(DNA) ]n dNTP] -Catalyzed by DNA polymerases -Incoming dNTP (dATP, dGTP, dCTP or dTTP) reacts with the DNA chain to form one new deoxyribonucleotide unit at a time with the release of a pyrophosphate ion -“Easy hydrolysis of PP iakes this reaction irreversible because the concentration of PP i is kept very small” The Flow of Information From RNA to DNA in Retroviruses Reverse TranscriptioTranscriptiTranslation • RNA à DNA à RNA à Protein • Catalyzed by reverse transcriptase, a viral enzyme -“On entering the cell, the RNA is copied into DNA through the action of reverse transcriptase” • Viral RNA à DNA-RNA hybrid à DNA transcript of viral RNA à Double-helical viral DNA • Viral RNA à Synthesis of complementary DNA strand à digestion of RNA à synthesis of second DNA strand RNA Molecules Involved in Gene Expression 1. Messenger RNA (mRNA) -“Template for protein synthesis, or translation” -Heterogeneous molecule -Third most abundant (rRNA, tRNA, mRNA) 2. Transfer RNA (tRNA) -Transports amino acids to the ribosome for protein synthesis -Second most abundant (rRNA, tRNA, mRNA) -About 75 nucleotides 3. Ribosomal RNA (rRNA) -Three different sedimentation coefficients -Most abundant (rRNA, tRNA, mRNA) -Catalyzes protein synthesis 4. Small Nuclear RNA (snRNA) -mRNA splicing 5. Small RNA molecule -Signal-recognition particle 6. Micro RNA (miRNA) -Inhibits the translation of mRNAs 7. Small interfering RNA (siRNA) 2 -Facilitates the cleavage of mRNA 8. “RNA is a component of telomerase, an enzyme that maintains the telomeres (ends) of chromosomes during DNA replication” 9. Primers -For DNA polymerase 10. Long non-coding RNA -Gene expression Transcription • DNA à RNA • (RNA) +nribonucleoside triphosphate ⇌ (RNA) n+1+ PP i Catalyzed by RNA polymerase • Factors required by RNA polymerases: 1. A template -Preferably double-stranded DNA 2. Activated precursors -Ribonucleoside triphosphates—ATP, GTP, UTP, and CTP 3. A div2+ent me2+l ion -Mg or Mn RNA Chain Elongation • Catalyzed by RNA polymerase -RNA polymerases do not need a primer to initiate synthesis • DNA synthesis by DNA polymerase vs. RNA synthesis by RNA polymerase Similarities: 1. “The 3’-OH group at the terminus of the growing chain makes a nucleophilic attack on the innermost phosphoryl group of the incoming nucleoside triphosphate” 2. “Driven forward by the hydrolysis of pyrophosphate” 3. Direction of synthesis: 5’ à 3’ Differences: 1. RNA polymerase does not need a primer to catalyze transcription 2. RNA polymerases are not as capable as DNA polymerases in correcting mistakes • The same RNA polymerase is responsible for synthesizing mRNA, tRNA, and rRNA in E. coli Complementarity Between mRNA and DNA • Figure 4.29 • The mRNA sequence (always written 5’ to 3’) is complementary to the DNA template sequence (written 3’ to 5’) from which it, the mRNA sequence, is synthesized • The mRNA sequence is identical to the coding strand of DNA • Codon -Sequence of three nucleotides -Specific for an amino acid -“Most amino acids are coded by more than one codon” -64 possible codons (20 amino acids + start + stop) 3 Promoter Sites for Transcription • Promoter sites -Regions in DNA template strands -The starting location of transcription -Region of template DNA strand to which the RNA polymerase initially binds -“Promoter sites lie upstream from the start site for transcription (+1)” • Prokaryotic promoter sites: 1. Pribnow box àTATAAT à-10 (10 nucleotides upstream from the transcription start site of +1) 2. -35 region àTTGACA • Eukaryotic promoter sites: 1. TATA box à TATAAA àAka Hogness box à -25 2. CAAT box à GGNCAATCT àSometimes present à -75 • “RNA polymerase initiation requires transcription factors to bind to the promoter sites and elongation requires additional factors” Termination of Transcription • Transcription proceeds until RNA polymerase synthesizes a terminator sequence • Terminator sequence -Encodes a termination signal • Termination signal in E. coli -Base-paired hairpin -The base-paired hairpin is an intrinsic terminator “formed by base pairing of self- complementary sequences that are rich in G and C” -Located at the end of mRNA coding regions • U aequence -Intrinsic terminator -Located at the end of mRNA coding regions -Adjacent U residues attached to the end of a base-paired hairpin • Rho -Protein responsible for Rho-dependent transcription termination • The termination of transcription is more complex in eukaryotes than in prokaryotes 4 Eukaryotic Post-Transcriptional Modification • Attachment of cap to 5’ end of mRNA -“During mRNA elongation, the 5’ end is modified by adding a non-coding 7-methyl-G via an inverted 5’-p-p-p-5’ linkage. Also, the first 1-3 coding nucleotides have 2’-OH à 2’-OMe” • Addition of poly(A) tail to 3’end of mRNA -Polyadenylation -Poly(A) tailà 100-200 A-residues • Benefits of post-transcriptional modifications: -Increases the life span of mRNA -The modifications help protect mRNA from attack by exonucleases -The modifications direct mRNA to the cytoplasm -The modification aid in the identification of mRNA by ribosomes • 5’- and 3’- untranslated regions (UTR) are common Transfer RNAs (tRNAs) • Responsible for carrying amino acids to ribosomes for protein synthesis • Adaptor molecules Structure of tRNA • Cloverleaf base-pairing structure • Two sites are present on a tRNA: 1. Amino acid attachment site à 3’ end of tRNA à CCA end 2. Template-recognition site à An anticodon that “recognizes a complementary sequence of three bases, called a codon, on mRNA” à The anticodon is recognized by its complementary codon “when both mRNA and tRNA are bound to the ribosome” • “Start and stop codons also occur with corresponding tRNAs” The Genetic Code • Major features of the genetic code: 1. “Three nucleotides encode an amino acid” 2. “The code is nonoverlapping” 3. “The sequence of bases is read sequentially from a fixed starting point, without punctuation” 4. “The genetic code is degenerate” -More than one code exists for most amino acids -“Degeneracy minimizes the deleterious effects of mutations” • Figure 4.5 the genetic code • Codons UUU and UUC specify Phe (the anticodons are AAA and GAA, respectively) • Codons UCU, UCC, UCA, UCG, AGU, and AGC specify Ser -Synonyms: “codons that specify the same amino acid” • AUG (start codon) à Met 5 Distinctive Codons of Human Mitochondria • The genetic code is almost but not completely universal in eukaryotes, prokaryotes, and archaea • The human mitochondria contains distinctive codons different from the standard genetic code -E.g. While UGA is a stop codon in the standard genetic code, UGA specifies Trp in the mitochondrial code -The distinctive codons of human mitochondria are a result of a distinct set of tRNAs encoded by mitochondrial DNA Initiation of Translation (Protein Synthesis) • In prokaryotes, translation is initiated with: 1. The recognition of the codon AUG (+1) or codon GUG by formylmethionine (fMet) carried by an initiator tRNA (fMet-tRNA) -fMet is the first amino acid -Uncharged N-terminus 2. The complementary base-pairing of a purine-rich sequence (-10) known as the Shine- Dalgarno sequence with ribosomal RNA *An Open Reading Frame (ORF) begins once both start signals are located • In eukaryotes, translation is initiated with: 1. AUG (+1) nearest the 5’ end of a mRNA with a 5’ cap -tRNA bound to methionine is the first amino acid -“At neutral pH, the N-terminus has NH -3????” Generation of Mature RNA • Newly synthesized RNA (pre-mRNA) contain both introns and exons -Exons: coding regions -Introns: non-coding regions • Introns are spliced out before translation occurs • Example of the generation of mRNA from pre-mRNA 1. Transcription, cap formation, and poly(A) addition of ????-globin gene 2. ????-globin RNA (1600 nt) obtained −This pre-mRNA contains both introns and exons 3. Splicing out of introns via spliceosomes -Spliceosomes: “assemblies of proteins and small RNA molecules” -“Introns nearly always begin with GU and end with an AG that is preceded by a pyrimidine-rich tract. This consensus sequence is part of the signal for splicing” 4. Generation of mRNA (900 nt) -The order in which the exons are located is the same in DNA and mRNA Exon Shuffling • “Introns were present in ancestral genes and were lost in the evolution of organisms that have become optimized for very rapid growth, such as prokaryotes” • Many exons encode functional proteins with a specific role such as ATP hydrolysis 6 • The shuffling of exons by DNA recombination can result in the formation of new genes and therefore increase diversity • Mating methods or retroviral infections can be used to generate novel exons Alternative Splicing • Alternative splicing is a method that can generate related proteins, or protein isoforms, “by splicing a nascent RNA transcript in different ways” E.g. the formation of a soluble antibody molecule from a precursor membrane-bound antibody molecule via alternative splicing -An antibody molecule is bound to a membrane via a membrane-anchoring unit encoded by a separate exon -“Alternative splicing of RNA excludes the membrane-anchoring domain” and leads to the generation of a soluble antibody molecule • “Alternative splicing is a facile means of forming a set of proteins that are variations of a basic motif according to a developmental program without requiring a gene for each protein” RNA Splicing • Powered by RNA catalysis • “No protein is required to splice out introns in Tetrahymena Thermophila (a protozoan) • “T. Thermophila self-splicing intron was used to show that mammalian splicing machinery can excise the same intron” Lecture 10/28: Exploring Genes I (p. 139-161) Major Biotechnology Techniques • Restriction-Enzyme Analysis • Blotting Techniques • DNA Sequencing • Solid-Phase Synthesis of Nucleic Acids • Polymerase Chain Reaction (PCR) Restriction Enzymes • Aka restriction endonucleases • Function: recognize and cleave specific DNA base sequences • Biological role: cleavage of foreign DNA • “Many restriction enzymes recognize specific sequences of four to eight base pairs and hydrolyze a phosphodiester bond in each strand in this region” -Type II and III restriction enzymes recognize specific cleavage sites Type II Restriction Enzymes • Capable of cleaving DNA without ATP • Characteristics of cleavage sites (recognition sites): -Twofold rotational symmetry 7 -Palindromic (inverted repeat) • Sticky ends or blunt ends (e.g. when cleaved by HaeIII) can result from cleavage The Separation of Restriction Fragments via Gel Electrophoressis • Restriction fragments: pieces of DNA produced with the cleavage of a DNA molecule by a restriction enzyme • Gels commonly used to separate DNA fragments: -Polyacrylamide gels à for the separation of DNA with roughly 10 to 1000 base pairs -Agarose gels à for the separation of DNA with roughly 50 to 20,000 base pairs • Increased cross-links in gel à slower DNA migration E.g. 20% polyacrylamide gel • Fewer cross-links in gel à faster DNA migration E.g. 2% agarose gel • Visualizing DNA within gel: --Autoradiography à for the visualization of radioactive DNA Ethidium bromide or Sybr green à chemicals used to stain DNA; DNA molecules bound to these chemicals fluoresce and enable detection of DNA Southern Blotting • Technique used for the identification of a DNA fragment containing a specific sequence • Southern blot protocol: 1. Separate a mixture of restriction fragments via gel electrophoresis 2. Transfer separated DNA fragments to a nitrocellulose membrane 3. Add 32P-labeled DNA probe to nitrocellulose membrane -“The probe hybridizes with a (target) restriction fragment having a complementary sequence” 4. Autoradiography -Autoradiography enables the detection of the DNA fragment of interest by revealing the location of the specific restriction fragment to which the probe was hybridized • Northern blotting à for RNA analysis Western blotting à for protein analysis via antibody probe Sanger Sequencing (Dideoxy Sequencing) • The controlled termination of replication • Protocol for four reaction mixtures (same procedure for all four): 1. Obtain single-stranded DNA to be sequenced 2. Prime synthesis with a primer 3. React with the following: 1. DNA polymerase I 2. Four radioactively labeled deoxyribonucleoside triphosphates (dATP, TTP, dCTP, dGTP) 3. Dideoxy analog of one of the nucleotides (“a different nucleotide for each reaction mixture”) -The dideoxy analog lacks a 3’-terminal OH group and therefore prevents the formation of a subsequent phosphodiester bond, terminating DNA replication 8 -Each dideoxy nucleotide inserts itself into the complementary DNA strand only at locations that correspond to a specific base in the original DNA strand being sequenced (e.g. “dideoxy analog of dATP will be inserted only where a T was located in the DNA being sequenced”) -Because dideoxy nucleotides can insert themselves at any location that aligns with a specific base in the original single strand, different fragments of complementary DNA strands are produced -DNA synthesis stops with the insertion of a dideoxy analog -A fluorescent dye is attached to each dideoxy analog (a different fluorescent colored dye for each dideoxy nucleotide) Capillary Electrophoresis • Technique in which fragments obtained from Sanger sequencing are passed through a narrow tube called a capillary • “As the DNA fragments emerge from the capillary, they are detected by their fluorescence” (via the fluorescent tag attached to each dideoxy nucleotide) -Different fluorescence à different wavelength -Different color à different base -High-resolution detection • “Single-nucleotide resolution affords direct sequence reads” • Common read-lengths à 100-600 nt Phosphite Triester Method • Solid-phase oligonucleotide synthesis • Figure 5.6 and Solid Phase Oligonucleotide Synthesis slide on lecture notes • Protocol: 1. Coupling of activated monomer with a growing chain -Activated monomer: nucleoside-5’-dimethoxytrityl-3’-diisopropyl-2-cyanoethyl- phosphoramidite (the 5’-OH group of the activated monomer is protected with dimethoxytrityl (DMT) and the 3’-phosphoryl group is protected by a beta-cyanoethyl (????CE) group) -Growing chain: the growing chain is linked to an insoluble support (e.g. resin) 2. Formation of phosphite triester intermediate 3. Oxidation by iodine of phosphite triester intermediate 4. Formation of phosphotriester intermediate 5. Deprotection of the DMT group on the 5’-OH group of growing chain by the addition of dichloroacetic acid -“The DNA chain is now elongated by one unit” 6. Repetition of steps 7. Removal of all protecting groups with NH 3 8. Release of newly synthesized oligonucleotide from solid support (resin) via addition of NH 3 • Usage of synthesized oligonucleotides: 32 -“A synthesized oligonucleotide labeled at one end with P or a fluorescent tag can be used (as a DNA probe) to search for a complementary sequence in a very long DNA molecule or even in a genome consisting of many chromosomes” 9 The Polymerase Chain Reaction (PCR) • Method used for the amplification of a given DNA sequence • Three steps for a single PCR cycle: 1. Strand separation -DNA strands are separated via heat (temperature increased to 95 deg. C) 2. Hybridization of primers -Abrupt cooling of solution (to 54 deg. C) enables the hybridization of the primers to a DNA strand 3. DNA synthesis -“The solution is then heated to 72 deg. C, the optimal temperature for heat-stable polymerases” (E.g. Taq DNA polymerase) -DNA polymerases elongate primers • The PCR involves multiple, single PCR cycles containing the three steps mentioned above • Figure 5.8 Review! Benefits of PCR • Assists in the identification of HIV • Assists in cancer detection • Useful for forensic evidence -Amplifying DNA obtained from bloodstains via the PCR Recombinant DNA Technology • Clone -“A collection of molecules or cells, all identical to an original molecule or cell” • Vector -DNA molecule capable of replicating within another cell -Used to transport foreign genetic information into another cell • Plasmids -“Naturally occurring circles of (double stranded) DNA that act as accessory chromosomes in bacteria” -Extrachromosomal DNA molecules -Capable of self-replication -Used as cloning vectors in E. coli—the most commonly used organism for cloning -“Plasmids replicate when E. coli replicates—plasmids are present in daughter cells Example of the Formation of Recombinant Molecules 1. Cleave circular plasmid pSC101 with restriction enzyme called EcoRI -Upon cleavage (opening up of curricular plasmid), complementary single-stranded ends (cohesive or sticky ends) are formed 2. Cleave any larger piece of DNA of interest with the same restriction enzyme, EcoRI -The single-stranded ends of the DNA fragments are now complementary to the sticky ends of the cut plasmid (via cleavage with a common restriction enzyme) 3. Join cut plasmid and DNA fragment at the sticky ends with DNA ligase 10 -Requirements for joining DNA molecules with a DNA Ligase to form recombinant DNA : 1. Free 3’-hydroxyl and 5’-phosphoryl group 2. “The chains joined by ligase must be in a double helix” + 3. ATP or NAD Formation of Cohesive Ends • Sticky ends (cohesive ends), required for the synthesis of recombinant molecules, can be generated by the following method: 1. Blunt-end ligation of a synthesized DNA linker to the ends of a DNA fragment or vector -The chemically synthesized DNA linker is capable of being cleaved by restriction enzymes -E.g. “The 5’ ends of a decameric linker and a DNA molecule are phosphorylated by polynucleotide kinase and then joined by the ligase from T4 phage” to form covalently-bound blunt-ended DNA molecules 2. Cleavage of chemically synthesized linker via a restriction enzyme to generate sticky ends -“Cohesive ends corresponding to a particular restriction enzyme can be added to virtually any DNA molecule” “Plasmids and Lambda Phage Are Choice Vectors for DNA Cloning in Bacteria” • Plasmids -Extrachromosomal DNA molecules -Circular -Capable of self-replication -Modified for the transfer of recombinant DNA into a target organism • Cloning vectors -Class of plasmids -Modified plasmids -Used to transport foreign genetic information into another cell -Properties of an efficient cloning vector: 1. Origin of replication 2. Selectable marker (e.g. antibiotic resistance gene) 3. Cloning site for the insertion of foreign DNA • Expression vectors -Class of plasmids -Modified plasmids -“In addition to antibiotic-resistance genes, (expression vectors) contain promoter sequences designed to drive the transcription of large amounts of a protein-coding DNA sequence” • Cloning vector and expression vector -Both plasmids commonly contain a polylinker • Polylinker -Region containing many different restriction sites so as to increase the potential number of restriction enzymes capable of cleaving the plasmid and, therefore, increase the number of possible DNA fragments that can be inserted into the plasmids 11 -E.g. the plasmid pUC18 contains a polylinker with restriction sites for 10 different restriction enzymes; interestingly, the polylinker is located within a region of the plasmid coding for the gene beta-galactosidase (lacZ gene); “insertion of a DNA fragment into one of the many restriction sites within this polylinker can be detected by the absence of beta-galactosidase activity” • ???? phage -Bacteriophage -Cloning vector -Alternative infection pathways: 1. Lytic pathway -“Lambda phage can multiply within a host and lyse it” 2. Lysogenic pathway -The phage DNA is integrated into the genome of the host cell and replicated along with the host-cell DNA -The replicated phage DNA remains inactive until an environmental trigger leads to its activation, followed by the generation of daughter viruses and subsequent lysis of the host cell • About 100 new virus particles containing the recombinant DNA are released upon the lysis of each E. coli cell • How to use a mutant ???? phage as a cloning vector: 1. Construct a mutant ???? phage -E.g. ????gt-???????? is a mutant ???? phage with only two EcoRI cleavage sites, compared to the five cleavage sites present in the normal phage 2. Remove the middle section of the mutant ???? phage after restriction digestion -The remaining two pieces of DNA make up 72% of the original genome length 3. Insert a long DNA segment between the first and last section of the mutant phage because “only DNA that is 75% to 105% of the length of wild-type ???? DNA (48 kb) will be packaged into new phage particles” 4. Conduct in-vitro packaging of the newly constructed recombinant molecule Bacterial and Yeast Artificial Chromosome Vectors • Bacterial and yeast artificial chromosome vectors are capable of accepting larger DNA inserts • Yeast artificial chromosomes -Contain the following: 1. Centromere 2. Autonomously replicating sequence 3. Pair of telomeres 4. Selectable marker genes 5. Cloning site -The centromere is involved in mitosis of the yeast cell -The telomeres can be extended without a template - DNA inserts cloned into yeast artificial chromosome vectors can range from 100 to 1000 kb The Creation of a Genomic Library in a ???? Phage 1. Fragmentation of genomic DNA by shearing or enzymatic digestion 2. Isolate fragments with lengths of about 15 kb via gel electrophoresis 3. Attach synthetic linkers to ends of fragments 4. Digest fragments with restriction enzymes to generate cohesive ends 12 5. Insert fragments into the middle section of the ???? genome -The ???? phage is a vector “prepared with the same cohesive ends” 6. “Propagate phage in E.coli and collect phage virions” -“The resulting lysate contains fragments of human DNA housed in a sufficiently large number of virus particles to ensure that nearly the entire genome is represented”—the genomic library is obtained • “For the human genome… a 99% probability of success requires screening about 500,000 clones” • The genomic library obtained can be used repeatedly so that the following gene- sequencing would not require a library • “PCR or Southern blot can identify clones with particular genes if some part of the gene sequence is known” Lecture 10/30: Exploring Genes II (p. 161-169) Probes Generated From a Protein Sequence • To isolate DNA from a large genome of DNA: 1. A genomic library must be available 2. A specific oligonucleotide probe for the gene of interest must be obtained • Methods for obtaining a specific probe: 1. The generation of a probe from a protein sequence of the protein encoded by the gene of interest -“Because a single peptide sequence can be encoded by a number of different oligonucleotides… peptide sequences containing tryptophan and methionine are preferred, because these amino acids are specified by a single codon, whereas other amino acids residues have between two and six codons” -For, say, a protein of seven amino acids, “256 distinct oligonucleotides (of a 21mer) mus16be synthesized to ensure that the probe matching the sequence is present” -“4 = 4 billion—any DNA that is a 16mer or larger is unlikely to occur by chance in the human genome” 2. Obtain a specific probe “from corresponding mRNA from cells in which it is abundant” Formation of a cDNA Duplex • Problem: bacteria cannot express mammalian genes which contain introns and exons because bacterial cells are incapable of splicing out introns • Solution: form a complementary DNA (cDNA) duplex from mRNA (mRNA does not contain introns) with the enzyme reverse transcriptase • The formation of a cDNA duplex: 1. Obtain a “DNA primer that is base-paired to the RNA and contains a free 3’-OH group” so that the enzyme reverse transcriptase can synthesize a DNA strand complementary to the mRNA strand, beginning synthesis at the primer -Primerà linked thymidine [oligo(T)] 2. Pair oligo(T) primer with poly(A) tail sequence located at the 3’ end of the mRNA strand 13 3. Reverse transcriptase begins the synthesis of the RNA-DNA hybrid in the presence of the four deoxyribonucleoside triphosphates 4. Increase the pH (add NaOH) for the alkali digestion of the mRNA template strand Attach oligo(dG) to the 3’ end of the cDNA strand via the enzyme terminal transferase 5. Begin synthesis of double-stranded cDNA with 1. DNA polymerase 2. Oligo (dC) primer or random primer 6. “Synthetic linkers can be added to this double-helical DNA for ligation to a suitable vector” 7. Insert vector containing cDNA into bacteria 8. Obtain cDNA library The Screening of cDNA Clones • Method for detecting cDNA (constructed from mRNA) expression in bacterial cells: 1. Construct an expression vector (a plasmid) containing 1. A bacterial promoter site and 2. A eukaryotic DNA insert (the cDNA) 2. Bacterial transformation of expression vector 3. Obtain bacterial colonies 4. Lyse bacteria to release the protein of interest 5. Transfer proteins to a nitrocellulose membrane 6. Incubate membrane with a radiolabeled antibody specific for the protein of interest 7. Use autoradiography to detect spots on film that indicate bacteria containing the protein of interest and therefore the cDNA Note: Quotations indicate text obtained directly from textbook or lecture notes References Berg, Jeremy, John Tymoczko, and Lubert Stryer. Biochemistry. 7th ed. W.H. Freeman, 2012. 1- 246. Print 14
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