Preparation of Plasmid DNA

Preparation of Plasmid DNA: A culture of cell containing plasmid grown in liquid medium is harvested and cell extract is prepared. Separation of plasmid from bacterial DNA is important. Plasmid is different from bacterial DNA in size and conformation. Both plasmid and host chromosome are circular but during preparation of extract the bacterial chromosome is always broken down into linear fragments. hence, can be separated on basis of linear or circular. Separation on basis of size: Lysed cell DNA are comparatively larger than plasmid DNA hence can be removed with cell debris by cetrifugation because bacterial chromosome is attached with cell envelope. Lysis is done carefully in presence of sucrose, treated with EDTA and lysozyme.Sucrose prevent from immediate cell burst. Instead sphearoplast are formed, which is cell with intact cytoplasm membrane and partialy degraded cell wall. To it,now ionic detergent TRiton-X100 is added for cell lysis. Ionic detergent cause chromosome breakage. Then centrifugation is done which leaves clear cell lysate. Lysate contain almost entire plasmid. Separation on basis of conformation: There are two types of DNA 1. Supercoiled or covalently closed circular DNA: During plasmid replication double helix of DNA is partially unwound with the help of topoisomerase, hence take the supercoiled conformation. This conformation is maintained if both strands are intact. 2. Open circular: If one of the polynucleotide strand is broken the double helix revert to relaxed state and plasmid takes open circular state. Supercoiled are easily seperated.

DNA extraction

Online Free life science study material. Hi More methods for extraction of total cell DNA: From grinding frozen plant cell or simple detergent treatment for animal cell we can get cell components. Plants and animals have carbohydrates etc other than bacterial cell. The plant carbohydrate are not removed by phenol extraction method. A detergent called CTAB (cetyl trimethyl ammonium bromide) forms insoluble complex with nucleic acid. This complex gets pecipitated leaving carbohydrates and protein and other contamination behind. This is centrifuged and resuspended in 1M NaCl which breakdown complex. Nucleic acid is now concentrated by ethanol and RNA removed by ribonuclease. Another method: Guanidium thiocynate has two properties 1. It denatures and dissolves all biochemical other than nucleic acid and can be used to release DNA from any type of cell or tissue. 2. In presence of Guanidium thiocyanate DNA binds tightly to silica plates. Silica placed in column and cell extract is added. DNA recovered by adding water due to destabilization of interaction between silica and DNA.

DNA purification

Online Free life science study material. Hi DNA Purification: There are three types of DNA Total cell DNA, Plasmid DNA and Phage DNA. For purifying Total Cell DNA: Brief summary: First we culture cells, the culture is then centrifuged to get pellet of cells. From the pellet cell extract is removed from which pure DNA is separated which is then concentrated for later use. DNA purification: Lysozyme enzyme is added to cell extract which digest polymeric compound that give rigidity to cell wall. For removal of lipid SDS is added. For removal of protein,protease is added for protein digestion and then phenol and chloroform in 1:1 ratio or only phenol is adde to precipitate protein. Ribonuclease is used to degrade RNA. Concentration of DNA: Organic extraction gives concentrated DNA, rest procedures give dilute DNA so to concentrate it following procedure is used. Ethanol is layered over DNA solution and DNA molecule is precipitated at interface. A glass rod is used to pull out long fibre. Alternatively ethanol is mixed with dilute DNA solution, the precipitate can be collected by centrifugation. This method leaves short chain of nucleic acid and RNA is lost at this stage. Measurement of DNA concentration: UV absorbance spectrophotometer are used to accurately measure DNA concentration. Amount of UV absorbed is directly proportional to amount of DNA A260(Absorbance at 260nm)of 1.0= 50 mg of double stranded DNA/ml. We can also check purity of DNA sample by UV spectrophotometer. If A260/A280=1.8 it means pure sample of DNA. if A260/A280 is less than 1.8 it means impurity of phenol or protein. Free life science studymaterial.

Recombinant DNA technology

Recombinant DNA technology. Hi Online Free life science study material. Plasmid: Plasmid circular DNA molecule present in procaryoytes.It is independant of main bacterial DNA. It is often used as selectable marker. It has one origin of replication sequence which makes it independent of main DNA. It has RP4 which carries gene for ampicillin, tetracyclin and kanamycin resistance. Size can vary from 1 kb to 250 kb. 10kb or less are used for cloning. Number can vary from 1-2 per cell (stringent plasmid) or 50 or more (relaxed plasmid)

Some plasmid carry tra gene which makes them conjugative plasmid. The bacteria having conjugative plasmid becomes conjugative. 5 Types of plasmid: 1. F or fertility: tra gene present. conjugal transfer of plasmid takes place in these. 2. R or resistance: Ampicillin, chloramphenicol etc resistance gene present. Makes bacterial cell resistant to the antibiotics. eg. RP4 3. Col plasmid: col gene for colicine is present. This protein kills other bacteria.eg colE1 of E.coli. 4. Degradative: this plasmid allows metabolism of unusual compound such as toluene, salicylic acid etc. eg. TUL plasmid of Pseudomonas putida. 5. Virulence: Vir gene is present (described in earlier blog). eg.Ti plasmid of Agrobacterium tumifaciens. M13 It is circular and entirely single stranded. It has 3 gene coding for capsid. It enters through pilus. It gets double stranded inside host. It does not integrate in host genome.Each daughter cell receives copy of M13 which replicate in them also. M13 vector is used in "Phage display". It is a technique for identifying pairs of genes whose protein product interact with each other. Free life science studymaterial.

RNA editing

Free life science studymaterial. Online Free life science study material. Hi RNA editing. This process is required to get variety of protein from single gene or DNA sequence. Any change such as addition, deletion or substitution in RNA can be achieved for getting desired protein from single gene. It always takes place in eukaryotes as procaryotes do not have introns. First observed in Trypanosoma gamibiense. Some times there is gap in DNA sequence it is also countere by gRNA or RNA editing mechanism. Mechanism pre mRNA is acted upon by guide/gi RNA. Editing is directional in 3'--5' direction. gRNA has stretch of A in between its sequence and this gRNA pairs with the targetRNA. The stretch of A does not pair with the target strand hence forms loop in between. Endonuclease acts on target RNA and causes a nick in target strand opposite to the AAAA of gRNA. (Consult diagram from book)After nick formation the loop in gRNA stretch and dUTP adds UUU opposite to AAAA in target RNA by thr help of TdT or terminal deoxynucleotidyl transferase. The gap is filled by ligase. The gRNA only adds UUU not any other nucleotide. Free life science studymaterial.

Elongation

Online Free life science study material. Hi Continue with translation.

Elongation: It requires Initiation complex, aminoacyl tRNAs, GTP and EF(elongation factors such as EF-TU, EFTs, EF-G) Now incoming aminoacyl tRNA bind to GTP bound EF-TU complex and this complex bind to A site. Eukaryotes do not have E site, tRNA removed directly from P site. Second aminoacid along with EF-Tu enters ribosome and bind to A site and amino acid 1 remains at P site, another next aminoacid binds to A site by shifting second to P site this way elongating the aminoacid chain. tRNA moves out from E site leaving aminoacid on chain. Each coming aminoacid requires peptidyl transferase enzyme for chain elongation. Peptidyl transferase step is energy independent and requires ribozyme. A peptide bond is formed between two amino acids by their tRNAs. Termination: 1. Takes place when ribosome encounters stop codon UAA,UAG,UGA. RFs are releasing factors, RF1 recognise UAA,UAG. RF2 recognise UAA and UGA. RF release the peptide, ribosome, mRNA, tRNA and the complex diassembles. some mutations: Missense mutation: cause aminoacid change. Nonsense mutation: change aminoacid to stop codon,cause termination of peptide. Silent Mutation: without apparent affect. Synonymous mutation: same aminoacid as parent, nucleotide change do not change amino acid. Neutral mutation: functionally equivalent aminoacid is replaced, not much effect. Free life science studymaterial.

Continue with translation. (Eucaryotes)

Continue with translation. (Eucaryotes) Online Free life science study material. Hi

Translation involves three organelles viz. Ribosome, mRNA and tRNA. Initiation, elongation and termination are three phases of translation. 1. Initiation: First step is tRNA charging. This takes place in cytosol. Similar step as in procaryotes. The difference lies in the initiation factors called eIFs. • The ribosome breaks before initiation. Smaller subunit 40S binds with eIF3 to form 40SN. eIF2 brings charged tRNA and binds to P site of ribosome and the complex becomes 43SN.In this step GDP is exchanged with GTP ,eIF2 is called GEF(guanine nucleotide exchange factor). • eI4F also called eIF4 helps in mRNA binding and forms 48SN complex. • 48SN bind with 60S sbunit to make 80S ribosome. • eIF1 causes stimulation of translation without eIF1 the 40S particle scan only few nucleotides. • eIF4A has helicase activity and unwinds any secondary structure of mRNA. • eIF4 is complex of eIF4A, eIF4E, eIF4G. adsbygoogle = window.adsbygoogle || []).push({}); • eIF4E has 5' Cap binding activity as ribosome has cap dependent entry and scans for AUG (start codon), eIF4G is adapter protein, capable of binding to eIF4E and 3'Poly A binding protein (PAB). In the next blog I will cover elongation.