Genetics Final Exam Condensed PDF

CHAPTER 10: GENE ISOLATION AND MANIPULATION Gene Cloning :extracting a gene of interest from an organism’s DNA and making copies (clones) of it Identify the gene: Locate the gene of interest. Enzymes for Gene Cloning: 1. DNA polymerase: Involved in DNA replication....

CHAPTER 10: GENE ISOLATION AND MANIPULATION Gene Cloning :extracting a gene of interest from an organism’s DNA and making copies (clones) of it Identify the gene: Locate the gene of interest. Enzymes for Gene Cloning: 1. DNA polymerase: Involved in DNA replication. 2. DNA pol I: For processing DNA fragments. 3. Ligase: Joins DNA fragments. 4. Restriction Enzymes (Molecular Scissors): Cut DNA at specific sequences 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 (like a plasmid) 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. Use ligase to join DNA fragments. Use a plasmid with an origin of replication to allow bacterial replication of the inserted gene. 2. Polymerase Chain Reaction(PCR):In vitro (outside cell) amplification w/ specific primers to replicate gene. Requires knowledge of the sequence surrounding the gene of interest. Restriction Enzymes & DNA Cutting Recognition of Palindromic Sequences: Sticky Ends: staggered cuts (EcoRI). Blunt Ends: even on both sides. EcoRI Example: ○ Cuts DNA to produce sticky ends (single-stranded overhangs). ○ ligate sticky ends of different DNA fragments to form recombinant DNA. Directionality: Using 2 diff restriction enzymes like EcoRI and XhoI can control direction of ligation. 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. Colony Screening: ○ Blue colony: No DNA insert (cleavage of X-gal). ○ White colony: DNA insert present (no cleavage of X-gal). Source of Thermostable Polymerases: 1. Discovery: In 1966, Thomas Brock found thermophiles in Yellowstone’s hot springs. 2. Thermophiles: Microorganisms with optimal growth at 60°C–108°C 3. 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) Inventor: 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. Initial PCR Steps: Heat to 95°C: Denature DNA to separate strands. Cool to Anneal: Allow primers to bind to target regions. Synthesize DNA: Taq polymerase extends the DNA strands. 2. Further Rounds of PCR: Continue cycles (~25–30 rounds) to exponentially amplify target DNA. 3. 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 bc digestion could cleave the fragment of interest 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 the opposite end of the target gene. 2. Set Up PCR:Denature, anneal, synthesize(multiple cycles to amplify the DNA) The forward primer will incorporate the T7 promoter into the DNA sequence at one end 3. Transcribe RNA: RNA polymerase will recognize T7 promoter and transcribe template into RNA Expressing Eukaryotic Genes in Bacteria Eukaryotic genes need to be converted into cDNA(complementary DNA) bc bacteria can’t process introns. Making cDNA: 1. Isolate mRNA: Extract mRNA from eukaryotic cells. 2. Reverse Transcription: Use reverse transcriptase (from viruses) to convert mRNA to cDNA. ○ Oligo-dT primers hybridize to the poly-A tail of mRNA.  ○ Reverse transcriptase synthesizes the cDNA strand. 3. Synthesize Double-Stranded cDNA: Remove mRNA & synthesize 2nd DNA strand using DNA polymerase I Producing cDNA Molecules with Sticky Ends 1. Add Sticky Ends to cDNA: Ligate oligonucleotide linkers containing EcoRI to the cDNA. 2. Insert into Vector: Use EcoRI to insert the cDNA into a plasmid vector. 3. Transcription: IPTG to inactivate Lac repressor so T7 pol can transcribe cDNA and express gene in bacteria. Purifying Protein: Histidine Tag (His-tag): Add sequence for 6 histidines to protein to purify w/ Ni2+-coated beads 1. Bind His-tagged proteins to Ni2+ beads. 2. Wash to remove unbound proteins. 3. Elute His-tagged protein using imidazole. Sequencing Genomes Fosmids and BACs (Bacterial Artificial Chromosomes): clone larger DNA fragments. human genome sequenced using large inserts to map the sequences across chromosomes. Modes of Delivering Recombinant DNA into Bacterial Cells: 1. Plasmids and BACs: Transformation (introduce DNA into bacteria). 2. Fosmids: Transduction (using phages to deliver DNA). 3. Bacteriophage Vectors: Infection followed by lysis to produce phage plaques. Confirming the Presence of the Insert 1. Grow Bacterial Colonies: Isolate colonies and test for plasmid presence. 2. Digest Plasmid with EcoRI: Confirm the presence of the gene insert by running on an agarose gel. 3. Hybridization (Southern Blot): Use a labeled probe to detect the DNA insert in colonies. Blotting Techniques: ○ Northern Blot: Detect RNA. ○ Southern Blot: Detect DNA. ○ Western Blot: Detect proteins. Restriction Enzyme-Independent Cloning Gibson Assembly: fast, cost-effective method for cloning multiple DNA fragments. ○ Can assemble multiple fragments in one reaction, no need for restriction enzymes. ○ Enzymes Used: 1. T5 Exonuclease: Chews back DNA ends to create overlaps. 2. DNA Polymerase: Fills in gaps. 3. DNA Ligase: Joins DNA fragments together. ○ Procedure: Mix fragments (including vector) and use enzymes to assemble into desired construct. Seamless Cloning: join DNA fragments in predetermined order w/out sequence restrictions or scars Synthetic biology (e.g., moving whole operons for metabolic engineering). Whole genome reconstructions.  ○ Ex. 1: 2015: >20 genes from plants, mammals, bacteria, yeast. ○ Ex.2: 2010: Mycoplasma laboratorium genome. Sequencing DNA: uses base-pair complementarity and DNA polymerase 5' to 3' activity. main methods Dideoxy Sequencing (Sanger Sequencing), developed by Fred Sanger. Applied to bacterial virus phiX174 (smallest genome). Uses dideoxynucleotide triphosphates ( w/ missing 3' OH group) to terminate chain elongation. Sanger Sequencing Steps ○ DNA template (PCR product, plasmid insert, genomic/cDNA). ○ Primer (3' OH for DNA synthesis). ○ Normal dNTPs (A, T, C, G). ○ A small amount of 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. Reading: DNA sequence read from the bottom (smallest fragment) to the top (longest fragment). ○ Fluorescent ddNTPs: Use of fluorescently labeled ddNTPs (green, red, blue, yellow for A, T, C, G). ○ Sequence read from electrophoresis, detected by a laser and recorded by computer. Genome Sequencing Process 1. Fragmenting Genome: Cut the genome into random fragments and sequence each fragment. 2. Overlap & Contigs: Overlap sequence reads to assemble contigs (continuous sequences). ○ Method developed by Craig Venter (founder of Celera Genomics, sequenced human genome). Whole Genome Shotgun Sequencing (WGS): Sequence first, map later. Paired-End Reads: ○ Sequence both ends of a fragment to help assemble larger genomes. ○ End reads from multiple clones overlap to create a full genome. Next Generation Sequencing (NGS) Example: Illumina dye sequencing (many systems available). Key Features: ○ Sequencing without cloning into microbial hosts. ○ Parallel sequencing of millions of DNA fragments. ○ Short reads (