MOLGENLEC: Molecular Tools

• Gel electrophoresis is used to separate different species of nucleic acid and protein.
• In DNA gel electrophoresis, melted agarose is poured into a form equipped with removable comb.
• The comb "teeth" form slots in the solidified agarose.
• DNA samples are placed in the slots.
• Sanger, Maxam, Gilbert developed two methods for determining the exact base sequence of a cloned piece of DNA.
• Ordering the fragments by size tells the base sequence of the DNA.
• The process of Sanger DNA sequencing yields a series of DNA fragments whose size is measured by electrophoresis.
• The last base in each fragment is known as that dideoxy nucleotide was used to terminate the reaction.
• Modern DNA sequencing is based on the Sanger method.
• The Sanger DNA sequencing method uses dideoxy nucleotides to terminate DNA synthesis.
• An electric current is run through the gel at a neutral pH.
• DNA is negatively charged due to phosphates in its backbone and moves to the anode, the positive pole.
• Small DNA pieces have little frictional drag so move rapidly.
• Large DNAs have more frictional drag so their mobility is slower.
• The result of DNA separation by agarose gel electrophoresis distributes DNA according to size, with the largest DNA pieces nearest the top and the smallest DNA pieces nearest the bottom.
• DNA is stained with a fluorescent dye.
• Comparison with standards permits size estimation in DNA gel electrophoresis.
• DNA hybridization is used in the selection of clone of interest in cDNA library construction.
• Health hazards associated with nucleic acid hybridization include increased sensitivity due to the use of a multiplier effect of an enzyme that is coupled to the probe for the molecule of interest.
• cDNA library construction is a simplified process that involves extracting mRNA, reverse transcription to make cDNA, and ligation in vector.
• Nucleic acid hybridization involves the ability of one single-stranded nucleic acid to form a double helix with another single strand of complementary base sequence.
• In Southern Blots, digests of genomic DNA are separated on agarose gel, the separated pieces are transferred to filter by diffusion, and the filter is treated with alkali to denature the DNA, resulting in ssDNA binding to the filter.
• Genomic library construction is a complex process that involves DNA extraction, digestion with restriction endonuclease, ligation in vector, and selection.
• Antibody screening is necessary in both cDNA library construction and genomic library construction.
• The mobility of fragments in DNA gel electrophoresis is plotted against concentration.
• The probe in Southern Blots is cDNA hybridizes and a band is generated corresponding to the DNA fragment of interest.
• Western Blots use a similar process to Southern blots, but with proteins instead of DNA.
• Techniques for isolated nucleic acids in nucleic acid hybridization include fluorescence in situ hybridization microscopy.
• Colony and plaque hybridization are techniques used in nucleic acid hybridization.
• Southern Blots are used in nucleic acid hybridization to identify specific DNA fragments.
• RT-PCR begins by converting mRNA to DNA, then uses a forward primer to convert single-stranded DNA to double-stranded DNA.
• The specificity of an antibody is determined by the epitope it recognizes.
• In real-time PCR, as DNA strands separate, anneal to forward and reverse primers, and to a fluorescent-tagged oligonucleotide complementary to part of one DNA strand.
• In Standard PCR, short pieces of DNA (primers) are added that hybridize to DNA sequences on either side of the piece of interest, causing initiation of DNA synthesis through that area.
• Polyacrylamide is usually used in protein electrophoresis.
• Antibody screening is a process to determine the specificity of an antibody.
• The selected region DNA now doubles in amount with each synthesis cycle.
• The preferred emulsion for Autoradiography is x-ray film.
• RT-PCR continues after standard PCR.
• The log of molecular weight (or number of base pairs) is used in electrophoresis of unknown DNA to estimate size.