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Fibrous Protein
Fibrous protein is a protein with an elongated shape. Typically one such as collagen or intermediate filament protein which is able to associate into long filamentous structure.

Colagen is a triple helix formed by three extended proteins that wrap around one another. Many rodlike collagen molecules are cross-linked together in the extracellular space to form collagen fibrils that have the tensile strength of steel. The striping on the collagen fibril is caused by regular repeating arrangement of the collagen molecules within the fibril.

Elastin polypeptide chains are cross-linked together to form rubberlike, elastic fibers. Each elastin molecule uncoils into a more extended conformation when the fiber is stretched and will recoil spontaneously as soon as the stretching force is relaxed.



Energy Flow Through Ecosystem
The diagram shows the sun light is a form of energy, provides one of the basic components needed by green plants in order to produce food in the form of sugar. This reaction occurs through photosynthesis by the special organs which are called chlorophyll of the plant. These plants, also referred to as producers, release a small amount of the energy they capture in the form of heat.The ATP is able to be made by cellular respiration.

Reference Source: Biology: Concepts & Connections with MasteringBiology (6th Edition) by Neil A. Campbell, Jane B. Reece, Martha R. Taylor and Eric J. Simon (Jun 9, 2010)



The Diels-Alder reaction
The Diels-Alder reaction is one of the most common reactions that is happened in organic chemistry. The Diels-Alder reaction is a cycloaddition reaction between a diene and a dienophile in order to produce a six-membered-ring. When the two carbon-carbon double bonds are positioned next to one another, a conjugated diene could be formed. A non- conjugated diene is a molecule that has two olefins which are not next to each other. Conjugated dienes undergo a cycloaddition reaction with certain double bonds to afford cyclohexenes and related compounds. The formation of new carbon-carbon bonds is one of the most important aspects of synthetic organic chemistry. When a synthetic sequence calls for the formation of a ring of carbon atoms, this problem is compounded. Fortunately, the formation of six-membered carbon rings is much simpler than it would first appear. This reaction normally forms a six-membered ring from two pieces: a conjugated "diene" and a "dienophile". The main requirements for these species are that the conjugated diene must be somewhat electron rich and able to achieve the s-cis conformation, and that the "dienophile" have a two-atom π system that is relatively electron poor. The mechanism of the reaction begins with the diene assuming the higher energy,but more reactive, cis conformation.

Reference Source: Diels-Alder - Endo and Exo - ChemTube3D, The Diels-Alder Reaction: The endo rule - people.bu.edu people



Biotechnology
The majority of specialized recombinant DNA molecules used in biotechnology have been constructed by subcloning procedures. Several hundred of these recombinant molecules are vectors which have been designed to meet specific needs in molecular biology and biomedical research. For example, some vectors have high copy numbers and will produce large amount of subcloned DNA inserts. Others have been designed to facilitate in-vitro transcription, super-expression of proteins in-vivo, DNA sequence analysis, replication in both prokaryotes and eukaryotes, facile conversion between double-stranded and single stranded forms of recombinant DNA, the cloning of large DNA fragments are single expression of characteristic marker genes which aid in the selection of cloned DNA. Subcloning involves the ligation of a previously cloned and purified DNA molecule into a vector. The resulting recombinant molecule is then introduced in the selection procedures are performed and the recombinant DNA is purified.

The kanr gene
Kanamycin is an aminoglycoside which interferes with translation by binding to the 70S prokaryotic ribosome. The drug interferes with translation by causing misreading of messenger RNA. The kanamycin resistance gene is form an E.coli transposon designated, Tn 903. A transposon is a segment of DNA that can become integrated at many different sites along a chromosome, especially a segment of bacterial DNA that can be translocated as a whole. Transposons are sometimes referred to as "jumping genes" for the ability to move from one chromosomal location to another within a genome or between genomes. The Tn 903 transposon encodes resistance to the antibiotics kanamycin and neomycin. The protein product of this gene is a 3'-aminoglycoside phosphotransferase which inactivate Kanamycin by covalent modification. The modified drug can not bind to the ribosomes. The cloned fragment encoding the gene is approximately 1300 base pairs in length and possesses Eco RI generated cohesive termini (sticky ends). Both the fragment and the recognition site are diagramed in the picture. The arrows in the Eco R1 recognition site diagram in the picture, indicate where Eco R1 cleaves this sequence. Eco R1 treatment results in staggered or "sticky" ends a protruding 5' phosphate on the adenine and a recessed 3' hydroxyl group on the guanine. The structural gene contains 813 bases which would code for a poly peptide consisting of 271 amino acid residues. The DNA fragment contains a promoter fir E.coli polymerase and has the required ribosomal binding sequences for mRNA transcripts.

Plasmid Vector
The vector is a 3000 base pair plasmid derived from the pUC series. The plasmid possesses a single recognition site for Eco R1 restriction endonuclease. The vector also contains several other unique restriction enzyme sites to facilitate the insertion of foreign DNA. The plasmid also contains an ampicillin resistance gene which codes for beta-lactamase (amp). The plasmid has been linearized with Eco R1 to produce termini compatible with the kanr fragment. This will facilitate the cloning of the kanr fragment into the plasmid.

Ligation
The ligation of the 1300 base pair kanr fragment to the linearized vector will be accomplished by the addition of T4 ligase to a buffered mixture of the two DNAs. T4 DNA ligase can be found in several E.coli strains. The T4 ligase catalyzes the fragmation of a phosphodiester bond by the condensation of a 5' phosphate and 3' hydroxyl group of adjacent nucleotides of the fragment and the vector. The enzyme is purified from T4 phage infected E.coli. It requires magnesium and ATP. Each phosphodiester bond formation results in the hydrolysis of ATP. The catalytic efficiency of the enzyme is optimal at 37°C. However, ligation of DNA fragments having cohesive termini is usually done at temperatures of 4°C to 22°C. Lower temperatures allow for annealing between complementary ends of DNA which is a prerequisite for the ligation of cohesive termini. The process is diagramed in picture below. In the simplest and most ideal case, the ligation of a one to one complex between vector and kanamycin insert would result in a circular recombinant plasmid consisting of 4300 base pairs. Phosphodiester bond formationwould occur between the guanine 3' hydroxyl group and the adenine 5' phosphate in the Eco R1 termini.

Reference Source: Biology 230 Lab Manual Grossmont College (2009)



Polymerase Chain Reaction
Polymerase Chain Reaction is known as PCR, which was invented in 1984 by Kary Mullis who was awarded a Nobel Prize for his work in 1994. Many utility of PCR are based on the ability to amlify DNA which is very important in lot of fiels. The PCR process uses an enzyme known as Taqpolymerase. This enzyme is purified from a bacterium originally isolated from hot spring and is stable at very high temperatures. Also ubcluded in the PCR reaction mixture are two synthetic oligonucleotides, known as primers, and the extracted DNA template also known as the target DNA. As the figure on right side shows, in the first step of the PCR reaction, the target complimentary DNA strands are melted at 94°C, when the Tq polymerase remains stable. In the second step f this reaction, known as annealing, the sample is cooled to 65°C to allow hybridization of the two primers to the two strands of the target DNA. In the third step of this reaction, the temperature is raised to 72°C and the Tq polymerase adds nucleotides to the primers to synthesize the new complementary strands. These three steps, which are denaturation, annealing, and DNA synthesis, constitute one PCR cycle. This process is typicality repeated for 30-40 cycles, amplifying the target sequence exponentially. PCR is performed in a thermal cycler, which is programmed to rapidly heat, cool and maintain samples at designated temperatures for varying amount of time. Reference Source: Biology 230 Lab Manual Grossmont College



Introduction
DNA fingerprinting is a recently developed method which allows for the identification of the source of unknown DNA samples. This method is very useful to determine the source of unknown DNA in criminal case and paternity, because it can provide more evidence. In contrast to the more conventional methodologies, such as blood typing, which can only exclude a suspect, DNA fingerprinting can provide positive identification with great accuracy. DNA fingerprinting involves the electrophoretic analysis of DNA fragment sizes generated by restriction enzymes. Restriction enzymes are endonucleases that catalyze the cleavage of the phosphate bonds within both strands of DNA. They require Mg2+ for activity and generate a 5 prime (5') phosphate and a 3 prime (3') hydroxyl group at the point of cleavage. The distinguishing feature of restriction enzymes is that they only cut at very specific sequences of bases called recognition sites. Restriction enzymes are produced by many different species of bacteria (including blue-green algae). More than 1500 restriction enzymes have been discovered. Restriction enzymes are named after the organism from which they are isolated. This is done by using the first letter of the genus followed by the first two letters of the species. Only certain strains or sub-strains of a particular species may be a producer of restriction enzymes. The type of strain or sub-strain sometimes follows the species designation in the name. A roman numeral is always used to show that the one out of possibly several different restriction enzymes produced by the same organism or by different sub-strains of the same strain.

Restriction Enzyme
Restriction enzymes are able to recognize specific double stranded sequences in DNA. Most recognition sites are normally range between 4 to 8 base pairs in its length. Cleavage occurs within the site. The cleavage positions are indicated by arrows in this figure. Recognition sites are frequently symmetrical, both DNA strands in the site have the same base sequence when read 5' to 3'. Such sequences are called palindromes. It is at such sites that restriction enzymes cut DNA. The size of the DNA fragments generated by restriction enzyme cleavage depends on the distance between the recognition sites. In general, the longer the DNA molecule, the greater the probability that a given recognition site will occur. The DNA of an average human chromosome is very large, containing over 100 million base pairs. A restriction enzyme has a 6 base pair recognition site, such as Eco RI, which would be expected to cut human DNA into approximately 750,000 different fragments. In order to determine the number of times a restriction enzyme cleaves double stranded DNA we use the equation in the figure.

No two individuals have the same pattern of restriction enzyme recognition sites. There are several causes for this fact. A large number of alleles exist in the population. These alleles are alternate forms of a gene. These result in alternative expressions of genetic trait. Chromosomes occur in matching pair, one of maternal and the other of paternal origin. The two copies of a gene at a given chromosomal locus constitute an individual's unique genotype. It follows that alleles have differences in their base sequences which consequently creates differences in the distribution and frequencies of restriction enzyme recognition sites. Other differences in base sequences between individuals can occur because of mutations and deletions. Such changes can also crate or eliminate a recognition site. The example in the picture shows how a silent mutation can eliminate a recognition site but leave a protein product unchanged.

Individual variations in the distances between recognition sites in chromosomal DNA are often caused by intervening repetitive base sequences. Repetitive sequences constitute a large fraction of the mammalian genome and have no known genetic function. However, changes in the number and position of these sequences effect the expression of neighboring genes. These sequences can occur between genes or are adjacent to them. They are also found within introns. Ten to fifteen percent of mammalian DNA consists of repeated, short sequences of bases in tandem arrays. The length of these arrays varies between individuals at different chromosomal loci. An example of a repeated sequence array in shown in the box below. The number of repeats varies from individual.

When these arrays are flanked by recognition sites, the length of the repeat will be able to determine the size of the restriction enzyme fragment generated. Variations in the length of these fragments between different individuals, in a population, are known as restriction fragment length polymorphisms which is also known as RFLP. RFLP are a manifestation of the unique molecular genetic profile, or "fingerprint", of an individual's DNA. These are several types of these short, repetitive sequences that have been cloned and purified.

Analysis of Complex DNA
There are two types of probes that commonly used for identification of genes. The single-locus probes that are called SLPs are able to detect a single segment of the repetitive DNA located at a specific site on a single chromosome. This probes have a result in one or two DNA bands corresponding to one or both chromosomes. If the segments recognized on the chromosome pairs are the same, then there will be one band. On the other hand, if they are different, it will appear as two bands. Several SLPs are available. Since more than one person can exhibit the same exact pattern for a specific SLP, several SLPs are used for a single test. Multiple-locus problem (MLPS) detect multiple repetitive DNA segments located on many chromosomes yielding 20 or 30 bands. It is because of the multi-band patterns, the chances of two people chosen at random exhibiting the same pattern is enormously remote. RFLP analysis of complex DNA, is facilitated by Southern Blot Hybridization, which is named after its discovered, Edward M. Southern. After electrophoresis, the DNA fragments are randomly depurinated by soaking the gel in acid. This reduces the binding between the strands and predisposes the deoxyribose-phosphate backbpne to cleavage by alkali. The DNA fragments are then denatured and partially broken by soaking the gel in alkali. This procedure causes the double stranded restriction fragments to be converted into single stranded form. A replica of the electrophoretic pattern of DNA fragments in the gel is made by transferring to a membrane of treated nylon. This is done by placing the nylon membrane on the gel after electrophoresis and transferring the fragments to the membrane by capillary action or suction by vacuum. The DNA becomes permanently adsorbed to the membrane, which can be manipulated much more easily than the gel. At this point the DNA is not visible on the nylon membrane. Analysis of the transferred DNA is often done by hybridization with a radioactive DNA probe. On the other hands, a non-isonic detection system can be employed to detect DNA bound to the sheet. In RFLP analysis, the probe is a DNA fragment that contains base sequences which are complementary to the variable arrays of tandemly repeated sequences found in the human chromosomes. Because these probes are chemically synthesized or cloned and puridied, it is relatively easy to label with them radioactive isotopes. A solution containing the single-stranded form of the probe is incubated with the membrane containing the tranderred, single-stranded DNA fragments. Under the proper conditions, the probe will only base pair to those fragments containing the complementary repeated sequences. The membrane is then washed to remove excess probe and is exposed to a sheet of x-ray film. Only those DNA fragments that have hybridized to the probe will reveal their positions on the film, because the localized areas of radioactivity cause exposure of the x-ray film. This process is known as auto-radiography. The hybridized fragments appear as discrete bands on the film and are in the same relative positions as they were in the agarose gel after electrophoresis. The reason that well-defined bands can now be visualized is because only a small fraction of the hundreds of thousands of fragments present contain sequences complementary to the probe. Only small amounts of DNA samples are required because the auto-radiography is very sensitive technique.

Identification of unknown DNA
The sample of an idealized banding pattern for a mother, child, father, and an unrelated are showed in the figure below. A child's DNA is a composite of its parent DNAs. Therefore, comparison of DNA fragmentation patterns can be obtained from the mother and child. It is going to give a partial match as a result. Bands in the child's fingerprint, which are not present in the mother's must have been contributed by the other. Because of allelic differences. It means that it will not appear all of the bands present in the parent's fingerprint in the child's fingerprint. However, the bands that do appear in the child's fingerprint must be found in either the father's or mother's print. In DNA fingerprinting laboratories, the two common restrict enzymes are called Hae III (GGCC) and Hinf I ( G'ANTC). They are 4-base and 5-base cutting enzymes respectively. The presentation of DNA analysis as evidence has become very important evidence in court cases such as murder, rape, and other types of crimes as well as in paternity determinations. In order to ensure greater accuracy, scientists incorporate standardization procedures in DNA analysis, such as determination of the exact size of individual DNA fragments in a DNA fingerprint.

Reference Sources: EDVOTEC