Final Biochem

• The first step is the formation of an enzyme-substrate complex, where the substrate binds to the active site on the enzyme.
• The most common method used for separating plasmid DNA from chromosomal DNA is the alkaline lysis method
• The fundamental distinction between chromosomal DNA and plasmid DNA is that plasmid DNA only contains extra genes that are not really necessary for the organism existence, but chromosomal DNA carries all of the information needed for the growth of the organism, development of the organism, and reproduction.
• Chromosomal DNA is highly supercoiled and compacted into nucleosomes, whereas plasmid DNA is less coiled and more relaxed.
• Plasmids can be easily isolated by using the alkaline lysis method because they have different physical properties than chromosomal DNA.
• Plasmids can be easily isolated by using different methods such as CsCl density gradient centrifugation or agarose gel electrophoresis.
• DNA ligase catalyzes the joining of two pieces of DNA together through phosphodiester bond formation.
• Salting-out is formally defined as the phenomenon when the solubility of a nonelectrolyte substance in water decreases with increasing salt concentration
• In salting out, the presence of high concentrations of salt causes an increase in the osmotic pressure of the solution, which leads to the precipitation of proteins from their solutions.
• DNA isolation methods include silica-based technology (DNA absorbs to silica beads/particles at a specific pH in presence of specific salts), magnetic separation (DNA binds reversibly to magnetic beads, which are coated with DNA-binding antibody), anion exchange technology, salting out, and cesium chloride density gradients.
• The process of isolating plasmid DNA involves breaking open cells, separating the nucleic acids from other cellular components, purifying the DNA, and removing contaminants.
• Proteinase K is added to digest any remaining protein that may interfere with subsequent steps.
• the peak absorbance for DNA is 260nm
• the peak absorbance for proteins is 280nm
• The type of bond that holds the phosphate group to the sugar in DNA's backbone is called a phosphodiester bond. Hydrogen bonds connect bases to one another and glycosidic bonds occur between deoxyribose groups and the base groups.
• Restriction endonucleases are enzymes that recognise DNA sequences, scan the sequence and cleave the fragment around or within that sequence. Exonucleases are enzymes that cleave the polynucleotide sequence either from the 5' end or the 3' end, one at a time. 
• a palindrome is a word or sentence that reads the same forward and backward.
• In PCR, primers bind to specific regions of the target DNA strands, which serve as starting points for synthesis of new DNA molecules. Each primer has a region that matches the template DNA and a region that does not match (overhang). These overhanging regions allow the newly synthesized DNA strands to form hydrogen bonds with the original DNA strands, creating double-stranded DNA again.
• Adenine (A) pairs with thymine (T). Guanine (G) pairs with cytosine (C)
• A restriction enzyme that cuts the backbones of both strands at non-adjacent locations leaves a staggered cut, generating two overlapping sticky ends, while an enzyme that makes a straight cut (at locations directly across from each other on both strands) generates two blunt ends.
• electrophoresis takes DNA to positive pole because its negative
• We use electrophoresis buffer for two main reasons. First, since water is a poor conductor of electricity, we add charged ions to water which allows the current to flow through our gel. Second, the buffer keeps our samples at a biologically appropriate pH that preserves their charge or structure.
• The smaller the fragment, the faster it moves toward the positive end of the gel.
• If all fragments have the same length, they will migrate together and appear as one band.
• During electrophoresis, negatively charged DNA fragments move towards the positively charged electrode due to the electric field generated between the electrodes.
• When a gel is stained with a DNA-binding dye and placed under UV light, the DNA fragments will glow, allowing us to see the DNA present at different locations along the length of the gel.
• However, for every 1000 nucleotides inherited there is one site of variation, called polymorphism, in the population. These DNA polymorphisms induce the change in the length of the DNA fragments produced by digestion with restriction enzymes in the course of a fingerprinting technique.
• In molecular biology, restriction fragment length polymorphism is a technique that exploits variations in homologous DNA sequences, known as polymorphisms, populations, or species or to pinpoint the locations of genes within a sequence.
• Restriction fragment length polymorphism (RFLP) analysis involves cutting genomic DNA into small pieces using specific restriction enzymes, separating these fragments based on size through agarose gel electrophoresis, and then hybridizing them to labeled probes.
• Southern blot is a method used for detection and quantification of a specific DNA sequence in DNA samples. This method is used in molecular biology.
• Principle. Southern blotting is based on the principle of separation of DNA fragments by gel electrophoresis followed by the identification by labeled probe hybridization. The DNA fragments are separated based on their size and charge during electrophoresis.
• A probe is a single-stranded sequence of DNA or RNA used to search for its complementary sequence in a sample genome. The probe is placed into contact with the sample under conditions that allow the probe sequence to hybridize with its complementary sequence.
• Variable number of tandem repeats (VNTR) is a location in DNA where a short nucleotide sequence is organized as a tandem repeat with variations in length between individuals.
• Short Tandem Repeat (STR): A type of VNTR consisting of repeats of two to six base pairs.
• PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA complementary to the offered template strand. Because DNA polymerase can add a nucleotide only onto a preexisting 3'-OH group, it needs a primer to which it can add the first nucleotide.
• The primers must be designed so they will anneal specifically to the target DNA at the beginning of the region to be amplified. Primers are usually synthetic oligonucleotides about 20 bases long
• In the next cycle, the newly synthesized strands serve as templates for further rounds of replication, resulting in exponential increase in the amount of DNA present.
• During PCR, the temperature is raised to denature the double helix, then lowered to permit annealing of the primers to the template DNA, and finally raised again to activate the DNA polymerase.
• In the first cycle, one copy of the gene is made from the original template; in the second cycle, two copies are produced because there were two templates available; in the third cycle, four copies are produced, etc.
• Generally, a complete polymerase chain reaction (PCR) requires five basic reagents, including DNA template, forward and reverse primers, DNA polymerase, deoxynucleotide triphosphates (dNTPs) and reaction buffer.