Extraction of DNA from Fresh Bone

Take a sample of nucleic acids like DNA from the bones to analyze gene expression in the search for somatic mutations in diseased tissues or other tumors, or to genotype archive material, when other sources of DNA are not available You can use a different kit that has been provided by biotechnology companies But if you want to extract a DNA sample from a large amount, you can use artificial methods, as described here to be cost effective


There are four procedures to ensure that the successful extraction of nucleic acids from the network:
1 disrupt the fabric so that the reagent for extraction can reach the cells

2 interference with the cell membrane so that the nucleic acids are released

3 nucleic acid separation from other cellular components

4 nucleic acid precipitation and volatilization

Material
1 DNA extraction buffer: Add 176 mol of 075 M sodium citrate, pH 70, 264 mol 10% sodium laurel seriously, and 250 g of guanidine isothiocyanate in 293 ml of distilled water and stir well Add 72 micro liters lyses buffer beta-mercaptoethanol/mL day usage
All chemicals must be capable of molecular biology Solutions can be stored at 4oC for 3 months

2 05 M ETDA: Add 9305 g EDTA to 300 ml of distilled water and add 10 N Noah, pH 80 Bring up to 500 ml Autoclave

Tries-EDTA: Add 1 ml of 1 M Tries to 200 micro liters 05 M EDTA To 100 ml with distilled water

3 3 M sodium acetate, pH 52: Add 4018 g sodium acetate per 800 ml of distilled water Adjust pH to 51 with glacial acetic acid Bring to 1 L with distilled water Autoclave

4 General reagents: Tries-saturated phenol pH 78 to 80 (Sigma), chloroform, ethanol 100% isopropanol

Method
1 Collect samples of bone in a sterile container containing buffered saline (PBS) and transport to the laboratory within 1-2 h
If DNA extraction is not started immediately, freeze the samples at-20oC or below for later use

2 Place bones in a clean glass dish plate With bone cutters or sharp scissors strong, isolated piece of bone about 1 cm3, and transfer it to bijoux 5 ml clean

3 Add 1 mol DNA extraction buffer and homogenize the tissue with scissors until a solution is obtained in mud

4 Transfer 500 micro liters spare screw cap conical mud in 15 mol Expender tubes

5 Add a volume of Tries-saturated phenol, followed by the volume of chloroform per tube Mix by inverting the tube several times or agitation Do not vortex, as long grooves cause the vortex-mixing of DNA cutting

6 Centrifuge tubes at 10 000 g for 20 minutes to separate phases

7 Transfer to the upper layer of fresh centrifuge tube (with respect to volume), taking care not to disturb the layer of milk on the interface Repeat steps 5-7 if the interface is disturbed

8 Add a volume of cold isopropanol and 01 volume of 3 M sodium acetate to the supernatant Stir well and let stand for 15 minutes on the ice

9 Centrifuge tubes at 10 000 g for 20 minutes to pellet DNA
East Expender tube to identify where is the DNA pellet Pellets will be visible at the bottom of the tube

10 Aspirate and discard supernatant, being careful not to disturb the sediment Wash the sample with 175 ml ice-cold ethanol and centrifuged at 10 000 g for 5 minutes Aspirate and discard supernatant and repeat wash

11 Dissolve the DNA pellet 10-50 micro liters of water or Tries-EDTA (you can pool the DNA from the sample at this stage) and measured by spectrophotometer or with Hoechst 33 258
Hoechst 33 258 is a DNA-specific dye that can be used to measure DNA

12 Store samples frozen at-20oC or lower 

DNA Double Helix

DNA macromolecules is a normal double helix Two polynucleotide chains, held together by weak thermodynamic forces, form the DNA molecule

Characteristics of the DNA double Helix

* Two strands of DNA forming a helical spiral, winding around the helix axis spiral right
* The two polynucleotide chains running in opposite directions
* The sugar-phosphate backbone of the two DNA strands wind around the helix as a fence line spiral stairs
* The bases of nucleotides in particular helix, stacked one above the other as staircases, spiral staircases

DNA Helix Axis

Helix axis is most obvious to look directly at the axis sugar-phosphate backbone on the outside of the spiral where the polar phosphate groups (red and yellow atoms) can interact with the polar environment Nitrogen (blue atoms) containing the base inside, stacking perpendicular to the propeller 

DNA damage

DNA damage, due to environmental factors and normal metabolic processes inside the cell, occurs at a rate of 1,000 to 1,000,000 molecular lesions per cell per day.While this constitutes only 0.000165% of the human genome's approximately 6 billion bases (3 billion base pairs), unrepaired lesions in critical genes (such as tumor suppressor genes) can impede a cell's ability to carry out its function and appreciably increase the likelihood of tumor formation.

The vast majority of DNA damage affects the primary structure of the double helix; that is, the bases themselves are chemically modified. These modifications can in turn disrupt the molecules' regular helical structure by introducing non-native chemical bonds or bulky adducts that do not fit in the standard double helix. Unlike proteins and RNA, DNA usually lacks tertiary structure and therefore damage or disturbance does not occur at that level. DNA is, however, supercoiled and wound around "packaging" proteins called histones (in eukaryotes), and both superstructures are vulnerable to the effects of DNA damage

Genetic Engineering

Methods have been developed to purify DNA from organisms, such as phenol-chloroform extraction and manipulate it in the laboratory, such as restriction digests and the polymerase chain reaction. Modern biology and biochemistry make intensive use of these techniques in recombinant DNA technology. Recombinant DNA is a man-made DNA sequence that has been assembled from other DNA sequences. They can be transformed into organisms in the form of plasmids or in the appropriate format, by using a viral vector.The genetically modified organisms produced can be used to produce products such as recombinant proteins, used in medical research, or be grown in agriculture.

Extraction of DNA from Fresh Bone

Take a sample of nucleic acids like DNA from the bones to analyze gene expression in the search for somatic mutations in diseased tissues or other tumors, or to genotype archive material, when other sources of DNA are not available You can use a different kit that has been provided by biotechnology companies But if you want to extract a DNA sample from a large amount, you can use artificial methods, as described here to be cost effective


There are four procedures to ensure that the successful extraction of nucleic acids from the network:
1 disrupt the fabric so that the reagent for extraction can reach the cells

2 interference with the cell membrane so that the nucleic acids are released

3 nucleic acid separation from other cellular components

4 nucleic acid precipitation and volatilization

Material
1 DNA extraction buffer: Add 176 mol of 075 M sodium citrate, pH 70, 264 mol 10% sodium laurel seriously, and 250 g of guanidine isothiocyanate in 293 ml of distilled water and stir well Add 72 micro liters lyses buffer beta-mercaptoethanol/mL day usage
All chemicals must be capable of molecular biology Solutions can be stored at 4oC for 3 months

2 05 M ETDA: Add 9305 g EDTA to 300 ml of distilled water and add 10 N Noah, pH 80 Bring up to 500 ml Autoclave

Tries-EDTA: Add 1 ml of 1 M Tries to 200 micro liters 05 M EDTA To 100 ml with distilled water

3 3 M sodium acetate, pH 52: Add 4018 g sodium acetate per 800 ml of distilled water Adjust pH to 51 with glacial acetic acid Bring to 1 L with distilled water Autoclave

4 General reagents: Tries-saturated phenol pH 78 to 80 (Sigma), chloroform, ethanol 100% isopropanol

Method
1 Collect samples of bone in a sterile container containing buffered saline (PBS) and transport to the laboratory within 1-2 h
If DNA extraction is not started immediately, freeze the samples at-20oC or below for later use

2 Place bones in a clean glass dish plate With bone cutters or sharp scissors strong, isolated piece of bone about 1 cm3, and transfer it to bijoux 5 ml clean

3 Add 1 mol DNA extraction buffer and homogenize the tissue with scissors until a solution is obtained in mud

4 Transfer 500 micro liters spare screw cap conical mud in 15 mol Expender tubes

5 Add a volume of Tries-saturated phenol, followed by the volume of chloroform per tube Mix by inverting the tube several times or agitation Do not vortex, as long grooves cause the vortex-mixing of DNA cutting

6 Centrifuge tubes at 10 000 g for 20 minutes to separate phases

7 Transfer to the upper layer of fresh centrifuge tube (with respect to volume), taking care not to disturb the layer of milk on the interface Repeat steps 5-7 if the interface is disturbed

8 Add a volume of cold isopropanol and 01 volume of 3 M sodium acetate to the supernatant Stir well and let stand for 15 minutes on the ice

9 Centrifuge tubes at 10 000 g for 20 minutes to pellet DNA
East Expender tube to identify where is the DNA pellet Pellets will be visible at the bottom of the tube

10 Aspirate and discard supernatant, being careful not to disturb the sediment Wash the sample with 175 ml ice-cold ethanol and centrifuged at 10 000 g for 5 minutes Aspirate and discard supernatant and repeat wash

11 Dissolve the DNA pellet 10-50 micro liters of water or Tries-EDTA (you can pool the DNA from the sample at this stage) and measured by spectrophotometer or with Hoechst 33 258
Hoechst 33 258 is a DNA-specific dye that can be used to measure DNA

12 Store samples frozen at-20oC or lower 

Components of DNA

DNA is a polymer monomer units of DNA nucleotides, and the polymer is known as a polynucleotide " Each nucleotide consists of five-carbon sugar (deoxyribose), a nitrogen-containing base attached to sugar and phosphate groups There are four types of nucleotides found in DNA, differing only in the nitrogen Four nucleotides are given a letter abbreviation as an abbreviation of the four bases
• A for adenine
• G is guanine
• C is for cytosine
• T thymine

DNA Backbone
DNA is a polymer chain with a sequence of alternating sugar-phosphate deoxyribose groups entered the second 3'-hydroxyl and 5'-phosphate hydroxyl groups in ester links, also known as "phosphodiester" bonds
DNA double helix

DNA macromolecule is a normal double helix Two polynucleotide chains, held together by weak thermodynamic forces, form the DNA molecule
Characteristics of the DNA double Helix
• Two strands of DNA forming a helical spiral, winding around the helix axis spiral right
• two polynucleotide chains running in opposite directions
• The sugar-phosphate backbone of the two DNA strands wind around the helix as a fence line spiral stairs
• Basics of specific nucleotides in helix, stacked one above the other like a spiral staircase 

Deoxyribonucleic acid

DNA, or deoxyribonucleic acid, is the hereditary material in humans and almost all other organisms Almost every cell in the body of someone having the same DNA Most DNA is found in the nucleus of the cell (which is called nuclear DNA), but small amounts of DNA can also be found in the mitochondria (where it is called mitochondrial DNA or medina)

The information in DNA is stored as a code which consists of four chemical bases: adenine (A), guanine (G), cytosine (C) and thymine (T) Human DNA contains about 3 billion bases, and more than 99 percent of their base is the same in all people The order or sequence of these bases determines the information available for building and maintaining an organism, similar to the way in which letters appear in a certain sequence to form words and sentences

DNA base pairs with each other, A with T and C with G, to form units called base pairs Each base is also linked to a molecule of sugar and phosphate molecules Together, the base, sugar and phosphate called nucleotides Nucleotides are arranged in two long grooves that form a spiral called a double helix Double helix structure is a bit 'like a ladder with the base pairs that make up the rung of the ladder and sugar and phosphate molecules forming the vertical arm of the scale

An important property of DNA that can replicate or make copies of itself Each double-stranded DNA can serve as a model for the duplication of DNA sequence This is important when cells divide because each new cell needs to have proper copies of DNA present in older cells 

Biochemistry-proteins

Proteins are linear polymers built from 20 different L-α-amino acids All amino acids are the general structural features including the carbon α to the amino group a carboxyl group and a variable side chain Only praline differs from this basic structure because it contains an unusual ring to the amino group of the N-end forcing half of the amide CO-NH in a side chain of amino acids conformation The fixed rules detailed in a list of standard amino acids has different chemical properties that produce three-dimensional structure of proteins and is therefore important for protein function Amino acids in the polypeptide chain linked by a peptide bond formed in a dehydration reaction Once connected in the chain of proteins called single amino acid residues and a circuit connected to carbon nitrogen and oxygen atoms known as a backbone main chain or protein peptide bond has two resonance forms that contribute some double bond character and inhibit rotation around its axis so that the carbon alpha approximately coplanar The other two dihedral angles in the peptide bond determine the form taken by the local backbone of proteins
Since the chemical structure of individual amino acids the protein chain has directionality End of the protein with a carboxyl group known as the free ends of carboxyl or C while the end with a free amino group known as the ends of N or amino acids
Proteins are generally used to refer to the complete biological molecule in a stable conformation while the peptide is generally reserved for short amino acid bloomers often lacking a stable three-dimensional However the boundary between the two is not well defined and is usually near 20-30 residues Polypeptide can refer to any linear chain of amino acids usually not long but often implies the absence of defined conformation

Benefits Microbiology

Robotics' refers to the beneficial live bacteria are consumed in small amounts to the potential health benefits See the pre-antibiotic substance consumed is determined to promote the growth of commensally bacteria in the body with the aim of improving Getting benefits on human health Or commensally bacteria outside the gut friendly bacteria can help in the digestion process In the US robotics is available as traditional dairy products equipped with the bacteria or as a food supplement in capsule form powder or tablet A special section related to the study of microbiology and the production of pro-biotic bacteria and pre-biotic

Synthesis of vitamins

Bacteria such as E coli present in human colon was involved in the synthesis of vitamins such as vitamin B12 folic acid biotin and potassium which can be used by the host This bacterium is often used for commercial preparations of vitamins such as riboflavin

The use of bacteria in milk and food industry

Bacteria particularly Lactobacillus lactic acid production is mainly used in
food preparation which involves the process of fermentation such as yogurt cheese bread fermented soy sauce pickles ketchup etc

The use of microbiology in agriculture and animal husbandry

Symbiotically biologically related bacteria able to convert atmospheric nitrogen into ammonia which contributes to enrich the soil and encourage the optimal growth of plants This bacterium is useful in agriculture and livestock and increase crop yields without using chemical fertilizers This bacterium is also important for the production of compost rotting organic material and serves as a rich source of nutrients for plants

Analysis of DNA or RNA

To measure the DNA content you can use a UV spectrophotometer with the benefits are not destructive and allows samples to be returned for further analysis or manipulation Spectrophotometer using the fact that there is a correlation between the absorption of ultraviolet light by DNA / RNA and its concentration in the sample In particular this post I will give you the facts about the relationship between DNA / RNA in the test wavelength spectrophotometer

1 The maximum absorption of DNA / RNA is about 260 nm This figure is the average absorption of the individual nucleotides that vary between 256 and 281 nm

2 In the case of RNA the concentration of a sample containing RNA may be calculated following equation:

40 x OD260 of sample = concentration of RNA (micrograms / mol)
And the equation for the concentration of DNA:

50 x OD260 = concentration of sample DNA (micrograms / mol)

Equations that describe the time-OD 260 is a concentration of RNA samples will be approximately 40 micrograms / ml (50 micrograms / ml for DNA)

3 We can also evaluate the degree of purity of nucleic acids by examining the absorption of other wavelengths where the protein and polysaccharide know the absorption maxima Proteins known to absorb strongly at 280 nm and polysaccharides can be identified with a maximum of up to 230 nm

4 Therefore in assessing the level of purity of nucleic acids using the relationship between measurements of the three wavelengths of 230 nm 260 nm and 280 nm

5 For example a sample containing only the following RNA extraction methods are not considered contaminated if the ratio is 1: 2: 1 and DNA is 1: 18: 1 (OD-230 reflects: 260: 280 ratio) If there are no significant deviations from the relationship then it is clear that the contaminants are present and further purification of the required samples
In many cases the purity and concentration can be further obscured by the presence of reagents used in the extraction process itself Some features are obvious in the scanning spectrophotometer which includes three wavelengths indicated Therefore when using spectrometry in the analysis of DNA or RNA you should be aware of potential problems that could produce a misleading report Also if the analysis reports and the concentration of DNA or RNA spectrophotometer are also necessary not only to obtain readings at 280 260 and 230 nm but also to analyze the whole range 200-320 nm Impressions reagents used in the extraction process can influence and provide misleading information that could influence the next manipulation

DNA sequencing and genomics

One of the most fundamental technologies developed to study genetics, DNA sequencing allows researchers to determine the sequence of nucleotides in DNA fragments Developed in 1977 by Frederick Sanger and coworkers, the sequence of chain termination is now routinely used for DNA fragments With this technology, researchers were able to study the molecular sequences associated with many human diseases

As sequencing has become cheaper, the researchers have been sequencing genomes of many organisms, using computational tools to put together a number of different fragments (a process called genome assembly) This technology is used to sequence the human genome, leading to the completion of the Human Genome Project in 2003 The new high-throughput sequencing technology dramatically lowering the cost of DNA sequencing, with many researchers hope to bring the human genome resequencing cost a thousand dollars

The large amount of sequence data available has created the field of genomics, research that uses computational tools to search and analyze patterns in the genome of an organism is full Genomics can also be regarded as a subfield of bioinformatics, which uses computational approaches to analyze large sets of biological data

Cell Signalling

Cell signalling and its perturbation in disease is perhaps the fastest growing area of biochemical research. Putting the term 'ceU signalling' into PubMed produces over 350,000 pubUshed papers. Therefore, there is need for an informative and readable introduction to the subject. Hancock attempts to do this, but I found the result somewhat disappointing.
The ceUs of multiceUular organisms need to communicate with each other in order to coordinate growth and differentiation. This is carried out by secretion of soluble signalling molecules that interact with plasma membrane receptors. These transduce the signal across the membrane and initiate a cytoplasmic signal cascade culminating in specific gene activation. There are thus three clear parts to the process, and mutations leading to changes in one or more stages of the process are evident for example in cancer cells. Unfortunately, the organisation of the book makes it difficult to understand the way in which these three stages interact.
The diagrams are of poor quakty and the figure legends are very brief and uninformative. For example, Figure 8.6 illustrates a complex signal pathway involving inositol substrates which I would think incomprehensible to a newcomer to the field. Common themes and structures are not emphasised; for example, activation of receptors or intracellular enzymes commonly occurs by phosphorylation of tyrosine, serine or threonine residues. Again, the book treats this topic in a superficial manner, and unifying principles such as src homology domains are not emphasised.
The many signalling pathways in the ceU converge on a few common intermediates and a smaller number of transcription factors. How does the cell know which signalling molecule has initiated the response and how do different pathways communicate (crosstalk) with each other? Again, these topics are not addressed in a meaningful manner.

Nuclear versus mitochondrial DNA damage

 

In human cells, and eukaryotic cells in general, DNA is found in two cellular locations - inside the nucleus and inside the mitochondria. Nuclear DNA (nDNA) exists as chromatin during non-replicative stages of the cell cycle and is condensed into aggregate structures known as chromosomes during cell division. In either state the DNA is highly compacted and wound up around bead-like proteins called histones. Whenever a cell needs to express the genetic information encoded in its nDNA the required chromosomal region is unravelled, genes located therein are expressed, and then the region is condensed back to its resting conformation. Mitochondrial DNA (mtDNA) is located inside mitochondria organelles, exists in multiple copies, and is also tightly associated with a number of proteins to form a complex known as the nucleoid. Inside mitochondria, reactive oxygen species (ROS), or free radicals, byproducts of the constant production of adenosine triphosphate (ATP) via oxidative phosphorylation, create a highly oxidative environment that is known to damage mtDNA. A critical enzyme in counteracting the toxicity of these species is superoxide dismutase, which is present in both the mitochondria and cytoplasm of eukaryotic cells.