Genetics Final Exam - Chapter 7: DNA Structure and Replication - PDF

GENETICS FINAL EXAM CHAPTER 7: DNA STRUCTURE AND REPLICATION Key Experiments Leading to DNA as Genetic Material 1. Frederick Griffith (1928) - Transformation in Bacteria in Streptococcus pneumoniae 2. Avery, MacLeod, and McCarty (1944)--Found DNA was a tra...

GENETICS FINAL EXAM CHAPTER 7: DNA STRUCTURE AND REPLICATION Key Experiments Leading to DNA as Genetic Material 1. Frederick Griffith (1928) - Transformation in Bacteria in Streptococcus pneumoniae 2. Avery, MacLeod, and McCarty (1944)--Found DNA was a transforming agent 3. Hershey and Chase (1952) - DNA entered the E. coli cells ○ 32P labels DNA., 35S labels protein. Experimental Evidence for Semiconservative Replication Meselson & Stahl (1958): Cesium chloride centrifugation: Used heavy nitrogen (N15) and light nitrogen (N14) to label DNA. DNA separated by density. 1st Gen: Intermediate density (one heavy and one light strand). 2nd Gen: A mix of light and intermediate density. Semiconservative DNA Replication—Arthur Kornberg used wrong polymerase(DNA pol I: too slow and short) DNA Polymerases in E. coli 1. DNA Polymerase I (PolA):Major repair enzyme. ○ 3’ to 5’ and 5’ to 3’ exonucleases 2. DNA Polymerase II (PolB):Replication restarts after damage. 3. DNA Polymerase III (PolC):Main replication enzyme, synthesizes long DNA strands. ○ 3’ to 5’ exonuclease activity 4. DNA Polymerase IV (DinB) and V (UmuD’2C): error-prone repair mechanisms. Replicating the Bacterial Chromosome: John Cairns incorporated 3H-thymidine into E. coli DNA to study DNA synthesis: ○ one round of replication: chromosome is a double-stranded circular DNA molecule. ○ 2nd round: DNA polymerase synthesizes DNA in two directions by both strands simultaneously Steps in DNA Replication(synthesis of lagging strand) 1. primase synthesizes RNA primer(5’-3’) to provide 3’ OH 2. DNA polymerase III elongates leading strand continuously 3. DNA synthesized in okazaki fragments, each starting with an RNA primer 4. DNA polymerase I cleaves RNA primers, fills in gaps, and synthesizes DNA(5’-3’ direction) 5. DNA ligase seals gap between okazaki fragments Replication Bubble in Prokaryotes (E. coli) OriC: The origin of replication in E. coli (circular genome). Replication in both directions from origin, forming two replication forks. Primosome: primase and helicase Replisome: Multi-protein complex that carries out DNA synthesis.  DNA Replication in Eukaryotes Multiple Origins of Replication S Phase DNA wrapped around histones and needs to be unwound Telomerase Telomeres & Telomerase Telomeres: protective repeated DNA sequences at ends of chromosomes RNA of telomerase: template for extending 3' overhang of telomeres by adding DNA sequence T-loop: 3' overhang of telomeres to stabilize chromosomes ○ Telomere Binding Proteins: TRF1 and TRF2 stabilize telomere and protect chromosome ends Elongation Process: telomerase extends telomere RNA moves along DNA, adding repeats to the 3' end Primase adds an RNA primer at the end DNA polymerase fills in the gap created by the primer DNA ligase seals the final gap Telomeres/Telomerase protecting ends discovery: Elizabeth Blackburn, Carol Greider, and Jack Szostak CHAPTER 8: RNA: TRANSCRIPTION, PROCESSING, AND DECAY Feature bacteria(prokaryotes) eukaryotes RNA synthesis and translation - occur simultaneously - RNA synthesis occurs in the nucleus - translation occurs in the cytoplasm Location of transcription - cytoplasm - nucleus Translation timing - ribosomes begin translating mRNA before - translation occurs after RNA processing and transcription finishes transport to the cytoplasm Cell complexity - less complex - more complex RNA processing - no processing required - 5’ capping, splicing, and 3’ polyadenylation Ribosome location - ribosomes are free in the cytoplasm - ribosomes free in the cytoplasm or attached to ER RNA vs DNA Feature RNA DNA Sugar Ribose(2’ OH) Deoxyribose(2’ H) Stability Less stable(single-stranded) More stable(double-stranded) Bases (U) replaces (T) Thymine(T) Methyl group replaced by hydrogen Methyl group at 5’ position Function Single-stranded Double helix structure can form secondary structures(hairpins) Makes it unstable(broken chain) Types of RNA Type Function mRNA Intermediary transferring genetic info from DNA to ribosomes for protein synthesis tRNA Transfers AAs to ribosomes for protein assembly rRNA Structural and catalytic components of ribosomes Most abundant RNA* snRNA Small nuclear RNAs involved in splicing and RNA processing miRNA Regulate mRNA stability and translation, linked to cancer siRNA Small interfering RNAs, involved in RNA interference piRNA Piwi-interacting RNAs, protect genome integrity lncRNA Long non-coding RNAs, involved in epigenetic regulation Ribozymes Catalytic RNAs capable of enzymatic activity CRISPR RNAs Guide RNA for genome editing RNA Synthesis Initiation AUG: The start codon for protein synthesis (first amino acid). -35 and -10 Sequences recognized by RNA polymerase. ○ distance between forms TATA box. RNA Synthesis and Transcription 1. Initiation: ○ Prokaryotes: core enzymes (α2, β, β', ω subunits) and a sigma factor (σ). Σ70 directs RNA pol. to promoter at -10 and -35, starting transcription at +1 site. After initiation, sigma factor dissociates, and RNA polymerase continues elongation. ○ Eukaryotes: Requires multiple transcription factors and promoter regions (e.g., TATA box). 2. Elongation: ○ RNA polymerase unwinds DNA and synthesizes RNA in 5′ to 3′ direction. ○ Nucleotides added to 3′ OH of growing RNA chain through nucleophilic attack by 5’ phosphate 3. Termination: ○ Terminator sequences (around 40 bp), rich in GC followed by A's, form hairpin structure in RNA.  ○ hairpin disrupts RNA-DNA so RNA polymerase pause, backtrack, and detach from DNA template ○ The U-A hybrid is weak, leading to the release of RNA and RNA polymerase. ○ 2nd mechanism: Rho factor binds RNA and unwinds DNA-RNA, dissociating RNA mRNA Decay(E.coli) 1. Pyrophosphohydrolase: Initial pyrophosphate removal 2. RNase E cleaves mRNA internally 3. exonuclease chews mRNA from 3’ end Miller Spread Technique: Visualize transcription under an electron microscope Prokaryotic vs. Eukaryotic Transcription Prokaryotes Eukaryotes Location Cytoplasm Nucleus mRNA processing No processing(mRNA naked) 5’ capping, polyadenylation, splincing mRNA export Immediately available for translation Must pass through nuclear pore to cytoplasm Translation simultaneously w/ transcription in cytoplasm Occurs in cytoplasm, after mRNA processing ribosomes Free in cytoplasm Free in cytoplasm or bound to ER Protein synthesis Immediately after transcription After mRNA export and processing Transcription Sigma factor guides RNA polymerase Promoters, enhancers, transcription factors regulation 1957 Volker & Astrachan: Showed RNA was the information transfer intermediate using radioactive uridine in a pulse-chase experiment. RNA Polymerases in Eukaryotes RNA Polymerase Location Function Involved in RNA Pol I nucleolus - Transcribed rRNA genes(except 5S) Ribosome synthesis RNA Pol II nucleus - Transcribes mRNA, long non-coding RNA, Transcription of most genes for microRNA, some snRNA protein synthesis RNA Pol III nucleus - transcribes small RNA genes Protein synthesis(translation) TATA Box: DNA unwinding point. Recognized by TATA Binding Protein (TBP) Binds DNA's minor groove using a β-sheet and distorts DNA structure to recruit other transcription factors. Saddle structure Transcription Initiation 1. Pre-Initiation Complex (PIC): 6 General Transcription Factors (GTFs) and RNA Polymerase II ○ TBP binds the TATA box, and GTFs guide RNA Polymerase II to the transcription start site. 2. TFIIH: causes RNA Polymerase II to start transcription and dissociates from most GTFs. ○ Helicase: Unwinds DNA. ○ Kinase: Phosphorylates RNA Polymerase II's C-terminal domain (CTD). Transcription Elongation CTD Phosphorylation: binding sites for other proteins involved in processing ○ Phosphorylation recruits enzymes for RNA processing. RNA Processing During Elongation: ○ 5’ Capping: Protects RNA from degradation, serves as a translation initiation site 1. Triphosphatase: takes away phosphate group from first nucleotide 2. Guanylytransferase: adds guanosine monophosphate 3. Methyltransferase: attaches methyl group to RNA strand to stabilize cap ○ Splicing: small nuclear RNAs (snRNAs). ○ Polyadenylation: Adds A residues to 3’ end, protected by Poly-A binding proteins. (Cleavage and Polyadenylation Specificity Factor) binds AAUAAA sequence. Cleavage by Poly-A Polymerase, followed by adenine addition. fold over to interact with proteins bound to cap structure Leaves complex behind sitting on TATA box waiting for other transcription factors to recycle off Stage Initiation - TATA box recruits TBP and GTFs to form the PIC - TFIIH phosphorylates RNA polymerase II for activation Elongation - RNA polymerase II phosphorylated CTD recruits processing enzymes Pausing - regulated by NELF, DSIF, and P-TEF-b to enable quick stress response - P-TEF-b phosphorylates RNA Polymerase II (CTD), resuming elongation Processing - co-transcriptional modifications: 5’ cap, splicing(intron removal), and 3’ poly A tail Termination - torpedo(Xrn2 exonuclease) or allosteric(conformational change) models release RNA pol II CHAPTER 9: PROTEINS AND THEIR SYNTHESIS Richard Roberts and Philip Sharp—Eukaryotic genes are discontinuous, containing exons and introns. Purpose of Introns: ○ Provide nucleotide triphosphates (via degradation). ○ regulatory roles (e.g., siRNAs). ○ Fold to create double stranded RNA after removed ○ Cleaved to become silencing RNAs Conserved Sequences in Intron Splicing Region Conserved Sequence Location 5’ Splice Donor GU Exon-intron boundary(5’) 3’ Splice Acceptor AG Intron-exon boundary (3’) Branch Point A 15-45 bp upstream of 3’ splice site Splicing Mechanism Two-Step Transesterification: ○ Step 1: 2’-OH of branch point A attacks 5’ splice site phosphate. Forms a 2’-5’ phosphodiester bond → intron lariat loop. ○ Step 2:3’-OH of exon 1 attacks the phosphate of exon 2. Exons join; lariat intron is released. Spliceosome Assembly Components: ○ 5 snRNAs: U1, U2, U4, U5, U6. ○ snRNPs and >100 proteins. U1 binds 5’ splice donor; U2 binds branch point A. RNA complementarity for precise binding. Protein folds RNA to bring exon boundaries together. removal of intron through reactive hydroxyls Self-Splicing Introns—Tom Cech (1989 Nobel Prize) guanosine cofactor for transesterification by providing reactive OH intro forms full circle before destroyed and brings reactants together Alternative Splicing: Produces multiple proteins from one gene by including/excluding different exons. Examples: 1. Rat α-tropomyosin: Tissue-specific exon selection. 2. FGFR2: Alternative isoforms of fibroblast growth factor receptors bind different ligands. Four Types: 1. Exon skipping (most common). 2. Alternative 3’ splice sites. 3. Alternative 5’ splice sites. 4. Mutually exclusive exons. mRNA Decay—Key Enzymes Deadenylase: Removes poly-A tail. Decapping Enzyme: Removes 5’ cap. Exonucleases degrade mRNA from 3’-5’ or 5’-3’. miRNA and siRNA: Gene Regulation Feature miRNA siRNA Mechanism Translation inhibitor RNA cleavage Complementarity with Target Imperfect Perfect Function Regulates many genes Targets specific mRNAs Processed from Processed from hairpin RNA by dicer Processed from long dsRNA miRNA Translation Inhibition Mechanisms 1. miRNA folds into a double-stranded structure 2. Dicer cleaves double-stranded RNA into ~22-nucleotide fragments. 3. RISC separates two strands of the miRNA fragment. 4. active miRNA guides RISC to a target by binding to (3’UTR) of the mRNA. 5. Action on Target mRNA: Degradation: RISC can degrade target mRNA if complementarity is high. Translation Repression: If complementarity is partial, translation of mRNA is repressed siRNA Pathway 1. Dicer cleaves dsRNA into ~21-22 nucleotide siRNA fragments. 2. RISC Complex: ○ Helicase creates single-stranded RNA. ○ Guide strand binds to target mRNA. ○ Argonaute (AGO) cleaves target mRNA. 3. Cleaved mRNA is degraded by exonucleases. RISC Models for Repression of miRNA Translation: 1. Interference with Translation Initiation - Cap Binding Block: no binding of translation initiation factors 2. Interference with Translation Initiation - Ribosome Assembly Block: blocks ribosome assembly at 5’ end 3. Interference with Elongation: physical block that halts elongation of polypeptide chain 4. Removal of the Poly-A Tail: RISC promotes deadenylation, leading to the removal of the poly-A tail. Three Experiments Demonstrating Gene Silencing 1. Fire and Mello (2004 Nobel Prize) – Muscle-specific unc-22 gene shut down by dsRNA.. 2. Jorgensen – transgene and plant's endogenous pigment gene were silenced due to dsRNA by transgene 3. Baulcombe – viral gene was transcribed into dsRNA, preventing virus replication Peptide Bond: b/ween amino end and carboxyl end of another, releasing 1 molecule of water Levels of Protein Structure Primary structure: Sequence of AAs (linear)  Secondary structure: ○ Alpha helix: 3.6 AAs per turn of the helix ○ Beta-pleated sheet: AAs in opposite directions, stabilized by hydrogen bonds Tertiary structure: disulfide bonds between cysteine residues Quaternary structure: Multiple polypeptide chains join(e.g., heme with alpha and beta subunits) rII Gene Mutation (Crick & Brenner) Proflavin mutation: mutation in rII gene of T4 phage causes it to grow only on one strain of E. coli Suppressor mutation: 2nd mutation can restore ability to grow on both strains Cracking the Code: Marshall Nirenberg and Others (1961) 1. Synthetic RNA Experiment: Used poly U RNA with E. coli extract in vitro ○ Produced polyphenylalanine peptide, identifying UUU as the codon for phenylalanine 2. Matched triplet codons to amino acids by analyzing resulting polypeptides 3. Created a codon table. 4. Tracked proteins synthesized from RNA using radioactive amino acids. tRNA Structure and Specificity tRNA structure: Each tRNA is specific to an AA ○ AA attachment site on 3’ end. ○ anticodon loop that recognizes corresponding codon ○ pseudouridine molecules: stabilize tRNA and used in mRNA vaccines for durability. Alexander Rich's: Identified tRNA structure. Wobble hypothesis: third nucleotide of anticodon can pair with multiple bases in the codon Wobble Base-Pairing Rules: flexibility in base-pairing 5’ End of Anticodon 3’ end of codon A U G C or U U A or G I(Inosine) A, C, or U Ribosome Structure and Function Ribosome Component Prokaryotic Ribosome Eukaryotic Ribosome Small subunit 16S rRNA + 21 proteins(30S) 18S rRNA + 33 proteins(40S) Large subunit 23s, 5s, 5.85 rRNA + 33 proteins(50S) 28S, 5S, 5.85S rRNA + 49 proteins(60S) Total ribosome size 70S(30S +50S) 80S(40S +60S)  RNA polymerase I: Synthesizes 18S, 28S, and 5.8S rRNA. RNA polymerase III: Synthesizes 5S rRNA. Ribosomal proteins translated in cytoplasm, then imported into the nucleus to assemble with rRNA. Key Ribosomal Binding Sites A site: Binds incoming aminoacyl-tRNA. P site: Holds growing peptide chain. E site: Holds tRNA ready to exit the ribosome. Protein Synthesis Overview—Key Components: Peptidyl Transferase: catalyzes peptide bonds, transferring growing polypeptide chain to next AA ○ in the 50S ribosomal subunit Decoding Center: ensures only tRNAs with matching anticodons for mRNA codon enter the A site. ○ in the 30S subunit Deacylated tRNA released from E site so ribosome moves to next codon Protein Synthesis Stages 1. Initiation of Translation Prokaryotes (Bacteria) Shine-Dalgarno Sequence:pairs with 16S rRNA, positioning AUG codon in P site in 5' UTR Initiation Factors (IF1, IF2, IF3): ○ IF1 & IF3: Prevent 50S subunit from associating with 30S subunit prematurely. ○ IF2: bring initiator tRNA to the P site. ○ Formyl-methionine (fMet): first AA added to initiator tRNA and placed in the P site. Eukaryotes Cap Binding Complex (eIF4A, eIF4B, eIF4G): Binds to mRNA cap structure. complex moves along mRNA in the 5’ to 3’ direction, searching for first AUG. When AUG is recognized, 60S ribosomal subunit joins 40S to form the 80S ribosome. 2. Elongation of the Polypeptide Chain 1. tRNA carrying an amino acid enters A site. 2. amino acid in A site forms a bond with growing peptide in the P site. 3. EF-G hydrolyzes GTP to GDP, shifting ribosome so tRNAs move from A → P → E sites. 4. Deacylated tRNA leaves through the E site. 3. Termination of Translation Stop Codons: UGA, UAA, UAG. Release Factors (RF1, RF2, RF3): Recognize stop codons and release polypeptide. Water Molecule enters peptidyl transferase center and triggers release of the polypeptide.  30S & 50S subunits separate, allowing 30S subunit to form a new initiation complex. Posttranslational Modifications Protein Folding: Many proteins require chaperones to fold correctly. Protease Cleavage: Inactive pre-proteins activated by cleavage Sugars added to proteins in ER and Golgi apparatus Phosphorylation: Addition of phosphate groups by kinases Ubiquitination: Targets proteins for degradation by 26S proteasome. Ubiquitin: 76-AA protein attach target proteins at lysine residues for their degradation. Extracellular Signals Altering Translation Kinase Signaling:Phosphorylation cascades alter transcription factors to regulate gene expression Regulation of mRNA: ○ Cap Removal: ○ miRNA ○ Pausing elongation ○ Poly A binding proteins removal Protein Localization—key mechanisms 1. Nuclear Localization—Nuclear Localization Signal (NLS) NLS recognized by cytoplasmic receptor proteins. Transport via nuclear pores 2. Signal Sequences for Organelle Targeting—signal sequence/peptide at N-terminal end. Proteins destined for membranes or organelles signal sequence directs the protein to appropriate channel for transport. Signal sequences cleaved by peptidase after transport initiation. 3. Secretion Pathway—N-terminal hydrophobic sequence. Hydrophobic signal sequence binds to proteins in the ER membrane. growing protein threaded into the ER lumen. 4. Processing in the Endoplasmic Reticulum (ER): Proteins are folded inside the ER. Ribosomes ensure proteins are synthesized directly into the ER lumen. 5. Golgi Apparatus: Proteins are packaged into lipid vesicles for their final destination. CHAPTER 10: GENE ISOLATION AND MANIPULATION Example: Cloning the Insulin Gene 1. Extract insulin gene from pancreatic cells. 2. Convert RNA to DNA. 3. Amplify DNA using PCR. 4. Cut DNA using restriction enzymes. 5. Insert DNA into a vector for cloning. 6. Introduce plasmid into bacteria for replication. Amplifying a Gene of Interest 1. Restriction Enzyme Cloning: Cut the gene with restriction enzymes, isolate it, and insert into a vector.. 2. Polymerase Chain Reaction(PCR):In vitro (outside cell) amplification w/ specific primers Requires knowledge of the sequence surrounding the gene of interest. Restriction Enzymes & DNA Cutting Recognition of Palindromic Sequences: EcoRI Example: ligate sticky ends of different DNA fragments to form recombinant DNA. Plasmids and Vectors—Types of Plasmids first Plasmids—Large plasmids used in gene cloning. Selectable Markers: ex. ampicillin resistance, to identify successful transformation in bacteria. Polylinker: A series of restriction enzyme sites used for easier insertion of DNA. Blue colony: No DNA insert (cleavage of X-gal). White colony: DNA insert present (no cleavage of X-gal). Source of Thermostable Polymerases:---Thomas Brock 1. Key Polymerases: critical for high-temperature PCR due to their thermostability ○ Taq Polymerase (Thermus aquaticus): Commonly used but error-prone. ○ Pfu Polymerase (Pyrococcus furiosus): Higher fidelity, processes larger DNA. Polymerase Chain Reaction (PCR)---Kary Mullis (1993 Nobel Prize). 1. Amplify target DNA region and add primers, dNTPs, and taq polymerase 2. Steps (repeated for amplification): Denature (95°C): Heat to separate DNA strands. Anneal (60°C): Cool to allow primers to bind to complementary sequences. Extend (72°C): Taq polymerase synthesizes new DNA strands.(optimal taq temp) 3. Cycle Repetition: Denature → Anneal → Extend repeated ~25 times. Amplification increases exponentially (~10⁶-fold after 25 cycles). Producing PCR Products with Sticky Ends 1. PCR 2. Digest PCR product with EcoRI: creating sticky ends for cloning. Ensure PCR primers include EcoRI recognition sites. ○ Verify target sequence doesn’t have EcoRI site Making RNA from Gene of Interest—in vitro transcription with T7 polymerase 1. Design Primers: forward primer containing T7 promoter sequence (18 bp: TAATACGACTCACTATAG) at 5' end followed by a sequence complementary to the target gene. reverse primer complementary to opposite end of the target gene.  2. PCR: forward primer will incorporate T7 promoter into the DNA sequence at one end 3. RNA polymerase will recognize T7 promoter and transcribe template into RNA Expressing Eukaryotic Genes in Bacteria—Making cDNA: 1. Extract mRNA from eukaryotic cells. 2. Oligo-dT primers hybridize to poly-A tail of mRNA. 3. Reverse transcriptase synthesizes the cDNA strand. 4. Remove mRNA & synthesize 2nd DNA strand using DNA polymerase I Producing cDNA Molecules with Sticky Ends 1. Add Sticky Ends: Ligate oligonucleotide linkers containing EcoRI to cDNA. 2. Use EcoRI to insert cDNA into a plasmid vector. 3. IPTG inactivate Lac repressor so T7 pol can transcribe cDNA and express gene in bacteria. Purifying Protein: 1. Bind His-tagged proteins to Ni2+ beads. 2. Wash to remove unbound proteins. 3. remove His-tagged protein using imidazole. Sequencing Genomes Fosmids and BACs (Bacterial Artificial Chromosomes): clone larger DNA fragments. Delivering Recombinant DNA into Bacterial Cells: 1. Plasmids and BACs: Transformation (DNA into bacteria). 2. Fosmids: Transduction ( phages to deliver DNA). 3. Bacteriophage Vectors: Infection followed by lysis to produce phage plaques. Confirming the Presence of the Insert 1. Isolate colonies and test for plasmid. 2. Digest Plasmid with EcoRI: Confirm presence of gene insert by running on an agarose gel. 3. labeled probe to detect DNA insert in colonies. ○ Northern Blot: Detect RNA. ○ Southern Blot: Detect DNA. ○ Western Blot: Detect proteins. Restriction Enzyme-Independent Cloning Gibson Assembly: fast, cheap method for cloning multiple fragments in 1 rxn, w/out restriction enzymes. 1. T5 Exonuclease: Chews back DNA ends to create overlaps. 2. DNA Polymerase: Fills in gaps. 3. DNA Ligase: Joins DNA fragments together. Seamless Cloning: join DNA fragments in predetermined order w/out sequence restrictions or scars Synthetic biology and Whole genome reconstructions. Sequencing DNA: base-pair complementarity and DNA polymerase 5' to 3' activity. Dideoxy Sequencing (Sanger Sequencing), developed by Fred Sanger. bacterial virus phiX174 (smallest genome). dideoxynucleotide triphosphates ( no 3' OH group) ○ DNA template (PCR product, plasmid insert, genomic/cDNA). ○ Primer (3' OH for DNA synthesis). ○ Normal dNTPs (A, T, C, G). ○ dideoxynucleotide (ddNTP: A, T, C, or G). 2. Reaction: ○ DNA synthesis with ddNTP incorporated at random, terminating chain elongation. ○ Separated by size on a gel (smaller fragments run further). 3. Fluorescent ddNTPs: fluorescently labeled ddNTPs (green, red, blue, yellow for A, T, C, G). Genome Sequencing Process—Cut the genome into random fragments and overlap sequence for contigs) - Craig Venter (founder of Celera Genomics, sequenced human genome). Whole Genome Shotgun Sequencing (WGS):Paired-End Reads:Sequence both ends of a fragment from multiple clones overlap to create a full genome. Next Generation Sequencing (NGS)---Illumina dye sequencing (many systems available). No cloning into microbial hosts. millions of DNA fragments. Short reads (