Main Ideas - DNA and RNA can be isolated from cells!- Restriction endonucleases= cutting DNA at specific recognition sites - Gel electrophoresis= separating nucleic acids according to size - Hybridization= detecting specfic DNA/RNA sequences - Polymerase chain rxn (PCR)= amplifying specific DNA sequences - Cloning= isolating and propagating single genes-->whole DNA libraries - Sequencing= determing the nucleotides sequence in DNA and RNA Lecture Objectives 1) Understand the molecular techniques discussed 2) Understand how combinations of the shown techniques are used in diagnostics and research DNA and RNA can be isolated from cells - Isolating genomic DNA from cells or tissues - Today we use kits mostly.!1) Lyse cells!- buffer at physiological pH, block DNases, detergent, proteinases!2) Purify DNA (phenol/chloroform extraction) !- pulls the DNA away from the protein, polar DNA goes into the water and protein is changed by the phenol (hydrophobic aa's on inside-->outside) so the protein avoids the water!3) Precipitate DNA (alcohol and salt), dehydrate the DNA!3) Resuspend DNA (H20 or buffer)!- Isolating RNA from cells or tissue!- Pretty much the exact same as DNA, use kits, only diff. is RNA is degraded much more easily !1) Lyse cells: physio pH, block RNases, detergent, proteinases, DNAases!2) Purify RNA (phenol/chloroform extraction)!3) Precipitate RNA (alcohol/salt)!4) Resuspend RNA (H20 or buffer)!!Restriction Enzymes: Cutting DNA into smaller pieces - Bacterial endonucleases (can cut in the middle of a double strand)!- Recog. short palindromic (can read the 5-3 seq. the same way on the strand below) DNA seqs!- Can gen. blunt or sticky DNA ends!- Type 1-2, most common type 2!- Uses:!- gene cloning and making DNA libraries!- often used w/southern blotting!- for RFLP= restriction fragment length polymorphism analysis (for pedigree analysis, prenatal diagnosis, and genetic fingerprinting)!!Gel Electrophoresis: Separating nucleic acids according to size - We've cut the DNA and now want to separate it by size!- Most common way to analyze DNA, RNA, and proteins!- Different gel types: agarose and polyacrylamind!- Since DNA/RNA backbone is negatively charged, in anelectric current, DNA/RNA moves toward the cathode (+ pole)!- Smaller molecules migrate faster than large one!!Hybridization= to identify a specifc DNA/RNA seq. in a large mix of DNA/RNA fragments!- Generate a sequence specific probe that's labeled radioactively or chemically (it's complementary to the target sequence).!- Two strands of a DNA double helix melt open (denaturation) upon hear (100c) or high pH (> or = 13)!- Complementary DNA single strands can re-anneal to form DNA double helices when temp or pH is lowered = Hybridization - need the right conditions (ex. not too cold) for the probe only binds to your target seq. and not other seqs!1) Run DNA/RNA on a gel to separate the diff. sized fragments!2) Denature DNA (-->ssDNA)-->transfer it to a membrane!3) Use hybridization methods w/a sequence specific probe!- DNA: Southern!- RNA: Northern !- Uses of Southern blotting:!- Identify a single gene and detect seqs in an organism!- analyze genetic patterns!- identify disease assoc. muts, deletions, duplications, and rearrangements!- identify related DNA seqs in the genome!- determined the # of copies of a specific DNA seq. (CNVs, VNTRs, DNA forensics)!- RFLP= restriction fragment length polymorphism Ex. Pedigree analysis!- looking at Sickle cell anemia: looking in a family for the presence of the sickle cell allele!- Only 1 NT change-->MstII enzyme recog. CCTNAGG-->can't recog. target seq. on Sickle allele!--> you get diff. sized fragments between the WT and the person w/sickle cell. Longer fragment= sickle cell allele, shorter fragment= WT allele !Ex. forensics and paternity test!!!Polymerase Chain Reaction (PCR): amplifying specific DNA sequences in vitro - Can use to isolate a gene and multiply it!- Taq DNA polymerase catalyzes PCR (can withstand high heat)!- Genomic PCR vs. RT-PCR!- RT-PCR: amplifying mRNA seqs in vitro!- isolate mRNA from sample (mature mRNA, no introns)!- add Oligo-dT primers (for polyA tail), anneal primer!- use reverse transcriptase to make the complementary cDNA strand!- add RNase H to digest the mRNA!- use thhe cDNA as a template in the regular PCR rxn, it will recognized by DNA polymerase and amplified!- Uses: amplify any DNA or mRNA seq. in our genome, close any DNA or mRNA seq, mutation analysis, analyze variable regions!!Cloning a gene/DNA segment - Need: Cloning vector (plasmid) + DNA segment of interest (insert)!- Take plasmid -->cut w/a restriction enzyme--> get blunt or sticky ends-->digest genomic DNA w/same restriction enzyme-->add DNA fragment to be cloned and it inserts w/same blunt or sticky ends-->covalently link w/DNA ligase-->recombinant DNA!- To amplify: take bacterial cell + ds recombinant plasmid DNA-->cell culture produces hundreds of millions of new bacteria-->many copies of purified recombinant plasmid isolated from lysed bacterial cells!- To clone a PCR product: !- use PCR primers with specific restriction enzymes (RE) sequences!- after PCR, digest PCR products w/RE!- digest target plasmid w/same REs and ligate!- To generate genomic DNA libraries!!!to generate cDNA libraries!DNA Library Uses:!- identify seq. of new gene (cDNA lib)!- id localization of new gene (genomic DNA lib)!- use cDNA clone to express protein!- determined which gene is mutated!- determine functionally relevant gene regions! !!!!!!!!!DNA sequencing (traditional): Determine the sequence of a DNA segment - Based on using dideoxyribonucleoside triphosphates (ddNTPs) to cause chain termination during a PCR like rxn w/one primer!- ddNTPs prevents strand extension at 3' end because they lack the 3' OH so you can't extend the DNA strand. When this ddNTP is incorporated-->immediate chain termination!- You had a ssDNA molecule to be sequenced + oligonucleotide primer for DNA poly + add many dNTPs (get incorporated in strand syn.) + small amt. of one type of ddNTP (ex. ddATP) + DNA polymerase!- each rxn has either ddATP, ddTTP, ddCTP, or ddGTP! - can read DNA seq. from bottom of gel!!Current approach: Sequencing using fluorescent-tagged ddNTPs - Each of the four ddNTPs is labeled w/a diff. fluor. dye that can be dtected by a laser-induced light emission (automated sequencer)!- Still same principle of chain termination!- Uses: identify muts in genomic DNA or cDNA, identify new gene sequences, confirm
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