module 1

• Chemical Method of DNA Sequencing involves fragmenting the DNA into manageable pieces, replacing the 5' end with 32P, and cleaving the DNA by base-specific chemical reactions such as G and A+G reaction, and C and C+T reactions.
• In the Sanger Method, DNA is fragmented into pieces, non-labelled ends are removed, and the pieces are separated using gel electrophoresis.
• Disadvantages of the Maxim Gilbert Method include radiation, UV light, time consuming, and expense.
• The Sanger Method requires Dideoxyribonucleotides (Only one kind), DNA Template, DNA Polymerase, and a 32P labelled primer.
• The Sanger Method involves attaching an oligonucleotide primer for RNA polymerase, adding dNTPs, occasional ddNTPs, assembling fragments of different lengths, separating by electrophoresis, and radiography.
• dNTP stands for deoxyribonucleoside triphosphate and ddNTP stands for dideoxyribonucleoside triphosphate.
• The Sanger Sequencing method can be improved by using fluorescent molecules instead of radioactive, automating reading, using a single reaction, and using automated cycle sequencing.
• Automated cycle sequencing involves DNA sample replication, primer for DNA polymerase to begin synthesizing new DNA strand, synthesis continues until a ddNTP is incorporated, DNA strands of various lengths beginning at same point, capillary and -ve electrode immersed into the tube, negative charges makes the fragments travel through the capillary, laser beams fired at passing molecules, and computer picks up the wavelengths and records.
• Pyrosequencing involves detection of pyrophosphate release and the generation of light on nucleotide incorporation.
• Enzymes used in pyrosequencing include Luciferin, Apyrase, DNA Polymerase, and ATP Sulfurylase.
• Steps of isolation of RNA include cell lysis, phase separation, RNA precipitation, RNA wash and redissolving, and measurement.
• Gene editing involves producing recombinant DNA by the steps: RE digestion of DNA plasmid, RE digestion of DNA insert, generating compatible sticky ends, purifying RE-digested plasmid and insert, and ligating them by DNA ligase.
• Guanidine Isothiocyanate is used to denature proteins.
• To "unstick" the cells from the dish, wash with PBS.
• Centrifugation is done after RNA starts precipitating.
• Isopropanol is added to precipitate the RNA.
• Differences between different restriction enzymes include optimal temperature, different pH, and ion composition of solution.
• Neoschizomers are bacteria that cut the same sequence in different locations.
• Restriction enzymes are used in gene editing and restriction mapping.
• Restriction mapping involves cutting the DNA with each enzyme singly, cutting the DNA with both enzymes, gel electrophoresis with DNA marker, visualizing the bands, determining the length of the DNA fragments, and generating the restriction map.
• Isoschizomers are different restriction enzymes that cleave the same DNA sequence.
• Chloroform or acidic phenol is added to separate phases of the lysate to separate phases of lysate.
• Bacteria use restriction enzymes to protect their DNA from restriction enzymes by methylating A or C nucleotides at locations where restriction enzyme would normally cleave.
• Restriction enzymes are used to cleave viral DNA.
• Restriction site is the site at which restriction enzyme cleaves the DNA.
• TRIzol is used to lyse cells.
• Restriction enzyme digestion produces blunt end, sticky end, or both.
• Apyrase function is the degradation of unused dNTPs.
• ATP sulfurylase takes released PPi and catalyzes reaction with APS to produce ATP.
• Agarose is good for separating 200bp to 50kb, and polyacrylamide is better for 10-200bp.
• If the DNA polymerase incorporates the dNTP into the chain, it releases pyrophosphate (PPi).
• ATP promotes the conversion of luciferin to oxyluciferin, which results in light being emitted.
• Loading dye/buffer contains glycerol to add density and help sample sink into the well, and one or two tracking dyes (bromophenol blue and xylene cyanol) to allow for monitoring of how far electrophoresis has proceeded.
• Ethidium Bromide intercalates with the DNA and emits fluorescence when excited by UV light.
• Light is detected and sequenced on a Pyrogram.
• If the dNTP is not complimentary, it will be degraded by apyrase.
• ATOP sulfurylase converts PPi and APS into ATP.
• Different DNA configurations migrate at different speeds, with Supercoiled DNA migrating the fastest, followed by linear and nicked DNA.
• Smaller beads carrying immobilized enzymes for pyrosequencing are deposited into each well.
• Reagents needed for gel electrophoresis include agarose or polyacrylamide gel, power back, buffer solution, loading dye, and Ethidium Bromide.