Genetics Objectives Chapter on Recombinant DNA Recombinant DNA is the term used to refer to a DNA molecule formed in the lab by joining together DNA sequences from different biological sources These are artificial creations not found in nature Recombinant DNA is also used to refer to the technology that is utilized to create and study these hybrid molecules Restriction enzymes are proteins used to generate specific DNA fragments These en zymes recognize and cut DNA molecules at specific nucleotide sequences They are used by bacterium as a defense mechanism against viral infections The restriction en zymes cut DNA into palindromic sequences where the base pairs are read the same but in the opposite direction of the complementary strand Restriction enzymes are used to clone DNA by obtaining a sequence of targeted DNA and allowing restriction enzymes to cut the DNA at recognition sites leaving sticky ends Also cutting bacterial vector with the same restriction enzymes is necessary to al low the recognition sequences to match When allowed to transform the vector takes up the segment and binds it to the cut sites on itself since they match DNA ligase re forms the phosphodiester bonds and the bacteria is now recombinant This bacteria al lowed to duplicate itself creates clones of the DNA sequence A genomic library contains cloned fragments of DNA isolated from chromosome The cDNA library contains cloned DNA copies of mRNA isolated from cells or tissues The difference between them allows them to be used differently When studying the whole genome or cloning the regulatory sequence the genomic library is used The cDNA li brary is used for isolating gene expression The process of converting mRNA to cDNA begins with the addition of oligo dT primer at the Poly A tail of mRNA Adding reverse transcriptase creates a double stranded du plex of RNA attached to DNA Adding partial amounts of RNase H digest portions of the RNA which creates small primers that can be converted to DNA by DNA poly 1 The fi nal step once DNA poly 1 creates the DNA is to add DNA ligase to seal the gaps and a double stranded cDNA is finished To identify specific genes in a clone probes have to be used to find them in the gene library This probe is used in a hybridization reaction by binding it to any complementary DNA sequence present To screen a library clones are grown on agar plates and al lowed to form colonies A replica is made of the colones onto a nitocellulose filter The filter is processd to lyse the bateriacells denature the dsDNA released from the cells into single strands and bind these strands to the filter The filter is screened by incuba tion with a labeled nucleic acid probe and the colonies that contain the clone can be de tected by x ray of the labeled probe Southern blot is a method that can be used to identify clones in a library containing a given DNA sequence and to characterize the size of the fragment also for creating re striction maps of segments within and near a gene Southern blotting has three compo nents separation of DNA fragments by gel electrophoresis transfer of the DNA to a ni trocellulose membrane and hybridization of the DNA fragments on the membrane using labeled probes Northern blot follows the same procedures as southern blotting except RNA is bound to the filter and this determines whether a gene is actively being ex pressed in a given cell or tissue type PCR is used to duplicated target segments of DNA It is done by having three steps including the denaturation of a DNA segment addition of a predetermined primer and lastly using heat stable DNA polymerase to replicate the strand This process is re peated for about 30 cycles and produces millions of copies of the target sequence To amplify a specific DNA sequences a primer has to be made of about 15 20 oligonu cleotides that are complementary to the 3 end of the denatured single stranded tem plates A thermostable DNA polymerase is necessary so that it can survive the heating stage in PCR when DNA is being denatured This heat stable DNA poly comes from Taq bacteria that is found in hot springs The principle used in dideoxy DNA sequencing is determining the exact sequence of nucleotides on the DNA sequence A DNA sequence is denatured and converted to single stranded The ssDNA is mixed with specific primers and the DNA is allowed to replicate in test tubes full of regular DNA nucleotides as well as some modified dideoxy DNA The dideoxy DNA stops the reading of the DNA and is also labeled to be later identified The theory is that as DNA synthesis takes place the polymerase occasionally inserts a dideoxynucleotide instead of a deoxynucleotide into a growing DNA strand Analyzing these strands shows stops at the dideoxy DNA and when compared to each other the position of the 4 bases and their order can be determined Today DNA se quencing is largely automated and computers can piece together the sequence of sev eral hundred thousand nucleotides Chapter on Genomics and Genome Sequencing Projects Bacteria genomes are small circular have very little repeating sequences lack in trons and are polycistronic Also the gene density of bacteria is really high Usually about one gene per kilo base of DNA which means a high portion of the DNA serves as coding DNA These shortness of the genome makes it easier to sequence In the eu karyote genome you find linear genes that are less densely packed monocistronic se quences with introns and a vast amount of the genome being repeated DNA During the human genome project the gaps around centromeres telomeres and repetitive se quences were skipped due to large amounts of repetitions in the sequences A DNA sequence can be identified by matching it to already known genes or compar ing them for high levels of similarities This process is called annotation and in today s level of Bioinformation relies heavily on Genomic databases stored on computers Using this process and sequencing the genes of many smaller organisms helps simplify the sequencing of human genes This is because the genetic code is universal and in eu karyotes many proteins are conserved and their sequences similar throughout organ isms Using the simple sequenced Genome of a mice or fly might identify certain se quences of a similar protein in the complex human genome A typical E Coli bacterium genome consist of 4 Mbp and 4 000 genes The eukaryotic unicellular yeast is 12Mbp and 6000 genes The Drosophila fruit fly has 180 Mbp and 16 000 genes And
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