Replication process

• DNA molecules are intercepted by a gyrase enzyme (topoisomerase) on either side. I cuts one of the strands of DNA and unwinds it, and mend it again creating two straight replication strands, while II cuts both strands, unwinds it and reseals it. Both prevent supercoiling
• The enzyme helicase helps gyrase by breaking the hydrogen bonds between the now-straightened strands on both sides, creating a replication bubble/fork from the origin of replication
• Primase is an enzyme which forms a strand of complimentary RNA bases, for the lagging strand, called a "primer".
• DNA polymerase III moves behind the Helicase enzyme and adds complimentary DNA free nucleotides to the 5' end forming Okazaki fragments of the growing strand using the RNA primer as a base. When everything is completed, the DNA polymerase returns to replace the RNA primer with DNA nucleotides as it has no need for a template
• Because DNA nucleotides (with the help of DNA polymerase III) get placed in the 3' direction, this continuous synthesis of the new strand is only applicable for the strand 3' to 5' end as its complimentary pair will be 5' to 3' end with nucleotides being added to the 3' end. This is the "leading strand" also called the "Watson" strand.
• The other strand, the "lagging" strand or "Crick" strand, is from the 5' to 3' end, meaning it's new complimentary strand is 3' to 5' end which does not conform to preferred synthesis orientation. In this case, uncontinuous synthesis occurs as new parts get added on as Okazaki fragments.
• Single-Stranded binding proteins (SSBs) is an enzyme that binds to the separated strands of DNA to stabilize them and prevent re-annealing of strands
• DNA replication is the production of exact copies of DNA with identical base sequences from two split parent strands
• Cells need to replicate DNA for growths in size, for reproduction, and to repair dead or damaged cells
• Cells replicate DNA for growth because after a certain cell size is reached, the Surface Area to Volume ratio cannot be sustained
• Conservative DNA replication suggests that the parents strands stay wound as they are and a completely new strand replicating it is made from scratch
• Semiconservative DNA (the most accurate description of our DNA) is the process whereby one new strand is made from each "parent" strand in the original DNA, making two exact, mixed copies.
• The Meselson-Stahl experiment proved that all DNA replications are of semi-conservative nature
• The Dispersive DNA replication model was suggested by Delbnuck and suggested that due to DNA's long nature, it's backbone was broken to create groups of 10 nucleotides to be copied without any unwinding
• Complimentary base pairing is essential to replication as it ensures proper copying of the sequence and prevents mutations
• A Polymerase Chain Reaction is undergone to copy specific parts of DNA over and over, usually for criminal investigation/forensic studies, paternity tests, or testing for viruses or diseases like COVID-19
• Polymerase Chain Reaction usually occurs in 30 cycles, creating over 1 billion copies of the original DNA segment with 3 sub-phases of cooling and heating
• Because of the heat-based thermal cycles of PCR, a special type of enzyme, called the Taq (Thermus aquaticus) polymer, is used because of its ability to withstand extremely hot temperatures without denaturing like other enzymes
• PCR do not need to occur in a cell and instead occur inside a buffer, most times
• The first sub-phase of PCR is the denaturation and breaking of the hydrogen bonds between the two parent strands of DNA which happens in 1 minute at 95 degrees celsius
• The second sub-phase of PCR is the Annealing process of cooling down the separated parents strands enough for primers to be able attach to its end and the Taq polymer being able to bind to it right behind the primer. This occurs for 1 minute at around 55 degrees celsius
• The third sub-phase of PCR is the Elongation as the Taq polymer attaches complimentary DNA nucleotides to finish the copied gene, occurring for 2 minutes at 72 degrees celsius
• The cycles of PCR occur as the three sub-phases repeat over and over with their new copies: each new copy separates with heat, and replicates upon cooling only to separate with heat again
• To PCR with RNA, the 'reverse transcriptase' enzyme is used to turn RNA into cDNA (complimentary DNA) for the process to occur.
• Gel electrophoresis is used to separate DNA molecules by length and size often used for criminal/forensic investigations and paternity tests
• Gel electrophoresis uses a gel called agarose as a molecular sieve to allow small molecules to pass further through the gel than larger/longer ones. Electric currents will pass through the gel often with buffer acting as electric conductors to force the negatively charged DNA to moved to the positive terminal of the electric current.
• for Gel electrophoresis to occur, DNA must be cut into pieces by restriction enzymes that act as molecular scissors, cutting out "restrictive regions" and only leaving core segments: repetitive segments of coding that are present in all human beings
• The "DNA Ladder" is a market with which the size of DNA fragments can be identified with the first lane being taken up to indicate such sizes
• DNA PCR should occur before any investigation to ensure there are multiples copies of the DNA to work with on the investigation
• Both PCR and Gel electrophoresis focus on non-coding DNA such as STRs (short tandem repeats) and VNTRs (variable number tandem repeats) that does not code for proteins, the latter more than the prior. Every human being has STRs and VNTRs but with the varying numbers and lengths of repeats being unique to each individual, allowing for a specific DNA fingerprint.
• Short Tandem Repeats, or STRs, have around 2-6 base pairs and sit very low on the gel in the DNA ladder.
• Variable Number Tandem Repeats, or VNTRs, can have around hundreds of base pairs and can sit anywhere near the middle or top of the gel DNA ladder
• Every person will have two alleles (alt versions) of their VNTR in a unique combination form their parents and can show variation in the amount of times the VNTR sequence is repeated
• STRs are loci (specific sites of DNA) where short and repetitive sequences are found and the number of these repeats are variable and can be compared. 1 STR is given directly from each parent which MIGHT have similar repeat lengths. Hence, they are the preferred method of creating genetic fingerprints- many are used.
• Homozygous genotypes are made of the same two alleles while Heterozygous genotypes are made of different alleles