One molecule, Many Forms: Why Uncut Plasmid DNA on Agarose Gel has 3 bands + the cut Plasmid DNA band on Agarose Gel

Rate of migration (from slowest to fastest):

Slowest Nicked plasmid > linear plasmid > supercoiled > circular, single-stranded Fastest

Nicked/Relaxed/Circular PLasmid: Definition of nicked plasmid: Nicked circle/relaxed circle. During extraction of plasmid DNA from the bacterial cell, one strand of the DNA becomes nicked. This relaxes the torsional strain needed to maintain supercoiling, producing the familiar form of plasmid. DNA found in the supercoiled form is not easily accessed by the replication machinery. During replication, cellular topoisomerases nick one strand of the DNA helix and relax the superhelical tension, thus allowing polymerases to gain access to the DNA. Using the rubber band analogy, nicked circular DNA is the rubber band without any twists introduced. This large floppy circle is the slowest migrating form in an agarose gel.

Linear: linearised DNA occurs when the DNA helix is cut in both strands at the same place. Linear DNA generally migrates between the nicked circle and the supercoiled forms. However, it may also migrate the same distance as the nicked circle -- it migrates as predicted by the length of the DNA (as compared to the molecular weight markers). Identify the linear DNA form on agarose gel by comparing uncut plasmid DNA with a sample of the plasmid that has been linearised using RE.

Supercoiled: native DNA conformation found in vivo and occurs when extra twists are introduced into the double helix strand. In the case of the DNA plasmid preps, this superhelical tension cannot be relieved because the ends of the plasmid are joined tgt. Supercoiled DNA migrates faster than predicted in an agarose gel due to its conformation. DEPENDING ON THE SITUATION: Supercoiled DNA is the desired species when isolating plasmid DNA.

Circular, single-stranded:

Definition of nicked plasmid: Nicked circle/relaxed circle During extraction of plasmid DNA from the bacterial cell, one strand of the DNA becomes nicked. This relaxes the torsional strain needed to maintain supercoiling, producing the familiar form of plasmid

issues with the presence of recA in the bacterial cell

In summary, open reading frames (ORFs) are defined as spans of DNA sequence between start and stop codons. Considered within a studied region of prokaryotic DNA sequence, only one of the six possible reading frames will be "open"

In eukaryotic genes with multiple exons, introns are removed and exons are joined together after transcription to yield final mRNA for protein translation

For gene finding in eukaryotes, the start-stop definition of an ORF only applies to spliced mRNAs, not genomic DNA, since introns may contain stop codons and/or cause shifts between reading frames.

ORF is a sequence that has a length divisible by 3 and is bound by stop codons, essentially, it is a series of DNA codons that does not contain any STOP codons, and furthermore, it is the specific reading frame, the 1 out of 3 options for the protein to be expressed correctly with the right amino acids.

All CDS are ORF but not ORFs are CDS.

CDS

• actual part of the gene which translates into a protein

ORF

• stretch of DNA btw a start codon and stop codon

• may contain introns

• can be part of the mRNA sequence

• may not be the whole coding region, especially in eukaryotes (could be just a single exon?)

In prokaryotes, CDS and ORF are the same

Both have a start and stop codon

They have a number of nucleotides which can be divided by 3

Once they translate, they produce aa

definition of CDS:

-consists of total exons of the gene and a start codon and a stop codon. It is the actual part of the gene that translates and produces the protein.

-does not contain 2 untranslated regions: 5' UTR and 3'' UTR

-introns are not included in the CDS

definition of ORF:

-nucleotide sequence located btw a start and a stop codon. There is no stop codon inside an ORF interrupting the genetic code which translates into a protein.

-there is no stop codon inside an ORF interrupting the genetic code which translates into a protein

-ORF includes a start codon, several codons in the middle region and a stop codon.

-interestingly, ORF has a length which can be divided by 3

In prokaryotes, CDS and ORF of a gene are the same

The sequencing method developed by Fred Sanger forms the basis of automated "cycle" sequencing reactions today. Fluorescent dyes are added to the reactions, and a laser within an automated DNA sequencing machine is used to analyze the DNA fragments produced.

Question for electropherogram; the yellow wavelength for guanine (G) is represented by black for legibility (definition: the quality of being clear enough to read, it is ambiguous but still high enough?). N is position at which the nucleotide cannot be determined (meaning that all the levels are too low such that none of the nucleotides can be determined for sure).

It is important to have nucleotides A,T,C,G and also to have dideoxynucleotides (ddNTPs), which are/acts as the fluorescent dyes and hence able to be recognized by a DNA sequencer

DEFINITION OF FIDELITY: The fidelity of a DNA polymerase is the result of accurate replication of a desired template

https://www.neb-online.de/wp-content/uploads/2015/04/NEB_Polymerase_Fidelity_Article_2012.pdf

isolated from different organisms

presence or absence of 3'-5' proof reading ability

high vs low fidelity

capability of amplifying longer or shorter DNA fragments

if the enzyme is inhibited by the presence of dUTP

Both Phusion and Taq polymerase enzymes are polymerizing enzymes that are capable of adding nucleotides to the 3’ free end of the DNA strand.

Both Phusion and Taq polymerases require a primer sequence to initiate polymerization.

Both Phusion and Taq polymerases are heat stable.

Both Phusion and Taq polymerases are used in PCR mechanisms to amplify DNA. When adding the polymerase to the reaction mixture, both Phusion and Taq polymerases are added last to ensure the efficiency of the enzyme.

Both Phusion and Taq polymerases are commercially synthesized for molecular biology experimental purposes.

Both Phusion and Taq polymerases require a cofactor to complete its function.

optimal temperature for best function

72-80 degree celsius

Characteristics of the polymerase

requres a co-factor, magnesium for its function

does not have the 3' -- 5' proofreading ability --> the error rate of Taq DNA polymerase is high in comparison to newer types of DNA polymerase (Eg. Phusion polymerase)

PRos of Taq polymerase

Taq DNA polymerase is still popular across the world of science due to the convenience and flexibility of the enzyme

manufactured by isolating the enzyme from Pyrococcus furiosus, which is an extremophilic Archaea

These microbes reside in extremely high-temperature conditions, thereby making the polymerase a highly heat-stable polymerase.

PRos of Phusion used to obtain extreme fidelity over the conventional thermostable Taq polymerase able to amplify long templates up to 7.5kb of genomic DNA optimal polymerisation capacity of Phusion is 72 degree celsius used in cloning products for sequencing, sequence analysis and mutational analysis has 3'--5' exonuclease activity allows proofreading of the newly synthesised strand after synthesis --> allows for easy repair of nucleotide mismatches --> lesser error rate

Overall advantages of Phusion polymerase are;

Extreme Fidelity

High Speed and reduced extension time

Robust Reactions and requires minimal optimization

High Yield

COns of Phusion

inhibited in the presence of deoxyuridine triphosphate (dUTP) --> when dUTPs are accumulated in the reaction mixture, it may inhibit the actions of the Phusion enzyme

Cloning: process of creating clones of organisms or copies of cells or DNA fragments while subcloning refers to a technique used to move a particular DNA sequence from a parent vector to a destination vector. Thus, this is the main difference between cloning and subcloning.