Chapter 6: analysis and characterization of nucleic Acid and proteins
Restriction enzyme and restriction enzyme mapping
Restriction enzymes are deprived from bacteria and other prokaryotes. It is an analysis that used in laboratories and bind to specific recognition sites. The recognition site usually has four through six base pairs. Restriction site mapping was first created using bacterial plasmids and it is used to analyze naturally occurring plasmids. The creation of restriction map involves DNA that has the use of restriction enzyme on it with a combination of other DNA that has the use of the enzyme. An enzyme PstI cuts a linear fragment of DNA. The DNA is incubated with an enzyme, Pstl, it can be separated using gel electrophoresis. BamHI enzyme can be used to determine the order of restriction fragments and recognize short sequences of DNA and splitting them at a target site. Restriction enzyme digestion causes patterns of fragments of the DNA. The patterns fragments in the DNA are used to determine and monitor the changes in size, structure and sequence of DNA. The number and location of a restriction site isn’t the same. It isn’t the same in human DNA because of inherited and somatic difference of the nucleotide. A molecular characteristic for DNA is the location and sequence of restriction enzyme sites. Restriction fragment length polymorphism (RFLP) is a method that analysis and cut from the same genetic group. RFLP is the resulting number and size of restriction fragments. This method is used to analysis the structure change in chromosomes that is associated with diseases.
Star activity means the relaxing and changes of the particular restriction enzyme that cleaves the DNA when reaction conditions is occurring. Star activity is in the restriction endonucleases category. Also, any kind of restriction endonucleases can cleave to non-canonical sites under extreme conditions. An increase of endonucleases cis known as star activity. High glycerol concentration, low ionic, high Ph, organic solvents, subtraction of magnesium with other cations. Star activity can be control with proper procedure steps like lowering the ph of the buffer to neural and using magnesium. Hind III can have the present of star activity if Mg 2+ is involved.
Blotting methods, especially Southern blot/ Comparison of blotting methods
For southern blot, the DNA is separated and cut with restriction enzymes. The fragments are isolated with gel electrophoresis then it depurated and denatured. After that it is transported to solid support like nitrocellulose. DNA fragments are exposed to a DNA or RNA labeled probes that specific in sequence to the region of interest. The first step is to digest the DNA with restriction enzymes and to find the correct enzyme for analysis. Typing of unknown organism or cloning will require the use several enzymes. For genomic DNA, 10 to 50 micrograms are used for each restriction enzyme for this analysis. Dependence of the sensitivity of the detection system, volume and configuration of wells and the number of target DNA, more or less of the DNA can be used. After, fragments of the DNA are resolved by gel electrophoresis. The size of the DNA region being analyzed is depended on by the percentage and nature of the gel. Target fragments are detected by hybridization single stranded DNA and RNA labeled detect marker. Double stranded fragments in the gel has to be denature and transferred to nitrocellulose membrane to have optimal hydrogen bonding of the probe and complementary sequences. Before the removal of the DNA fragments to membrane, double stranded DNA has to be denatured to single strands. This takes place when the DNA is still in the gel. Large fragments, greater than 500 base pairs can be efficiently denatured if they are depurated before. Large fragments are soaked in hydrochloride solutions which removes purine from the sugar phosphate backbone. This will help the large fragments denature completely. Sodium hydroxide denatures the DNA whiles it in the gel. Single stranded DNA binds tightly to the membrane for transferring.
Northern blot was created to analyze RNA structure and quantity. This technique is normally used to analyze the transcription of DNA and stability. It is also used to investigate the abnormalities of RNA, alternate splicing. Splicing abnormalities cause diseases like beta-thalassemia. Northerner blot indirectly shows the mutation of the regulatory or splicing signals in DNA. Since RNase can affect the RNA preparation, caution has to be taken into concern to keep an RNase-free environment. Efficient transfer of the RNA from the gel to the membrane requires completely denature. After the electrophoresis, the lanes can be cut out and soaked in ammonium acetate to remove the denaturant and stained with acridine orange or ethidium blue.
Western blotting is known as immunoblotting because antibody is used to target its antigen. This method is used to analyze the immunoassay in humans for HIV and hepatitis C virus. Electrophoresis separates the HIV proteins and transfer to bind to the nitrocellulose membrane. The serum from the patient is laid on the membrane and the antibodies will bind to the HIV proteins and to other corresponding proteins. Cell lysate or serum is separated using SDS-polyacrylamide gel or isoelectric focusing gel. The molecular weight and charge affect the migrations of the protein. Dithiothreitol and 2-mercaptoethanol are used to separate the proteins into proteins. Polyacrylamide concentration can be five percent to twenty percent, usually 1-50 micrograms of protein is pipette into each well. Before it is pipete in the well it has to be denatured with 1:1 with 0.04 M Tris HCI, Ph 6.8, 0.1% SDS.
Western and southwestern bot targets are proteins. Southern and northern blots probes are nucleic acid. The purpose of for northern and western blot is for gene expression. Southern and northern use fragments, after labeling and denaturing.
Stringency, what it is, calculation, variables that affect it
Stringency is a collection of condition where the target were exposed to probes. Very high stringency means that the probe will not bind to the target. Very low stringency will bind to unrelated targets which effect the analysis results, like a very high stringency. The temperate of hybridization affect stringency. Salt concentration of hybridization buffer and denaturants concentration affect the stringency also. The level of stringency is impacted by the nature of the probe sequence. High percentage of a probe for C and G bases will bind in a more stringent condition. An ideal hybridization condition can be calculated by the melting temperature. Melting temperature is represented as Tm. Tm can be represented as the amount of energy require to isolate the hybridized strand of the given sequence. Tm as a double stranded DNA has a formula: Tm=81.5C + 16.6 logM +0.41 (%G + C)-0.61 (%form amide) – (600/n). M=the sodium concentration in mole/L; n= #of base pairs in short duplex. RNA hybrids is consider to be more stable compared to DNA. RNA hybrids Tm=79.8C+18.5logM + 0.58(%G+C) +11.8(%G+C)-0.35 (%form amide)-(820/n). RNA hybrids have intermediate affinity: Tm=79.8C+18.5logM + 0.58(%G+C) +11.8(%G+C)-0.50 (%form amide)-(820/n). There’s a formula of Tm for short probes from 14-20 bases: Tm =4C * # of GC pairs + 2C * # of AT pairs
Probes and bybridization
Probes significance is to find one or more sequences of interest in a polymers of nucleic acid. Probes hybridize with target of DNA or RNA. The probe can be RNA or denatured DNA or any other modified nucleic acid. Fragments of DNA can be cloned on a bacterial plasmid and separated with restriction enzymes digestion and gel electrophoresis. DNA probes are made from the separation of sequence of interest from viral genomics and in vitro organic synthesis. Polymerase chain reaction synthesis DNA probes to make polymers of a particular sequence. The length of the probe determines the particular hybridization reaction. There are tens to thousands base pairs in the length of the probe. Transcription of DNA usually makes RNA probes. RNA probes are more sensitive with southern blot than DNA probes. Plasmid and DNA templates can synthesizes RNA probes. Coding RNA will hybridized to a DNA target that is double stranded. Complementary sequence are used for probes. A probe that has identical sequences at the target will not hybridize. Chemical methods have to synthesize locked nucleic acid and peptide nucleic acid. These types of nucleic acid are unaffected by nucleases. Structure that do not have negative charges can be hybridize easily. Protein probes are used for western blot for antibodies. Antibodies form a connection to specific immobilized target proteins. There are specify antibodies such as polyclonal and monoclonal. These antibodies are types of immunoglobulins that reacts against a particular antigen. In order to have a response for antigen, cells, nucleic acids, tissues and etc. have to be extracted. Monoclonal antibodies are hard to produce. However, Kohler and Milstein showed that spleen cells in immunized mice can be put together with myeloma cell from mouse to form hybridomas.
Be able to calculate Tm
Tm as a double stranded DNA has a formula: Tm=81.5C + 16.6 logM +0.41 (%G + C)-0.61 (%form amide) – (600/n).
RNA hybrids Tm=79.8C+18.5logM + 0.58(%G+C) +11.8(%G+C)-0.35 (%form amide)-(820/n)
RNA hybrids have intermediate affinity: Tm=79.8C+18.5logM + 0.58(%G+C) +11.8(%G+C)-0.50 (%form amide)-(820/n).
Formula of Tm for short probes from 14-20 bases: Tm =4C * # of GC pairs + 2C * # of AT pairs
Microarrays and chips
Microarrays came after macro arrays. DNA microarrays are used to measure macromolecule of genes. Over the years technology became more advance, such as spotting technology and the ability to deposit tiny targets spots on glass. DNA microarray, known as a DNA chip, is a glass slide that exhibit 384 spots on it. There can be up to a thousand targets that can be screen in a small area by miniaturizing the deposit of droplets. Microarrayers can put over 80,000 spots on a glass substrate. There was rough draft that was done with the microarrayer saying that human genome has less than 30,000 genomes. The glass microscope carries the array of target which is referred to as a chip. DNA, RNA, cDNAs, PCR products, and oligomers can be the target. There are spaces between the targets to avoid artifacts. DNA synthesis happens on glass or silicon to deposit targets on the chips. Proprietary photolithography is highly efficient for short oligomer on high-density oligonucleotide arrays. Over 100,000 targets can be put on the chip using this technique. Mutation analysis, single nucleotide polymorphism analysis and sequencing involves the use of high density oligonucleotide arrays. Microelectronic arrays is a method use to target a particular position on the array. The electrode has the array connected to it. The sample needs fluorescent labeling for analysis since microarrays and other high density arrays are read by automated fluorescent detection system. Expression arrays measures the transcript and protein production that close to a reference control that has been separated from normal specimens. Array CGH is a method used to determine what specific genomic loci that has deletions and amplification.