BIOL 282 CH 20: Recombinant DNA technology – Flashcards

Your essay should include an appreciated for the relative ease in which sections of DNA can be inserted into various vectors and the amplification and isolation of such DNA. You should include the possibilities of modifying recombinant molecules.

Recombinant DNA technology, also called genetic engineering or gene splicing, involved the creation of associations of DNA that are not typically found in nature. Particular enzymes, called *restriction endonucleases*, cut DNA at specific sites and often yield "sticky" ends for additional interaction with DNA molecules cut with the same class of enzyme. Isolated from bacteria, restriction enzymes fall into several classes, each having peculiarities as to structure and interaction with DNA. A vector may be a plasmid, bacteriophage, or cosmid that receives, through litigation, a piece or pieces of foreign DNA. The recombinant vector can transform (or transfect) a host cell (bacterium, yeast cell, etc.) and be amplified in number. In a DNA cloning experiment, DNA ligase is used to generate the covalent bonds of the phosphodiester backbone to yield an intact double-stranded DNA molecule. Restriction enzymes, on the other hand, break such bonds.

Even though the human gene coding for insulin contains a number of introns, a cDNA generated from insulin mRNA is free of introns. Plasmids containing insulin genes (from cDNA) are free of introns, so no processing issue surfaces.

Although bacteria are commonly used in cloning, other cell types are also very useful, such as yeast, mammalian, and so on. Bacteria are prokaryotes and as do not process transcripts as do eukaryotes; therefore, there is often an advantage to using a eukaryotic host

This segment contains the palindromic sequence of GGATCC, which is recognized by the restriction enzyme BamHI. The double-stranded sequence is the following. GGATCC CCTAGG

The simple answer to this question is to assume that one is asking about the advantage so the scientist of having restriction enzyme sites recognize palindromic sites. In this case, the answer would be that single-stranded overhanging ends are often generated which allow DNA from difference sources cut with the same restriction enzme to generate. complementary overhangs ,which can anneal to form reconbinant molecules, If one considers the question from a bacterial standpoint, the asnwer ism uch more involeved, In fact bacterial chromosomes

Plasmids were the first to be used as cloning vectors, and they are still routinley used to clone relatively small framgnets of DNA. Because of the tsmall size, they are relatively easy to separate from the host bacterial chromomes, and they have relatively few restriction sites. They can be engineered fairly easily (i.e. polylinkers and preporte gnes added). For cloning larger peices of DNA such as entire eularyotic genes, socmids are often used. For instance, when modifications are made int he baterial virus lambda, relatively large inserts of about 20 kb can be clones. This is an important advatnge when one needs to lond a large gene or generat a genomic library from a eukaryote I nadditiojn, some somids will aceept only inserts of a limited size, which means that smal, perhaps less meaningul fragments will not be cloned unnecessarily. Both plasmids and cosmids suffer from the limitation that thae can use onyl bacteria as hosts NBACs are artificual bacterial chromosomes that can be engineer for cerain qualities. YACs (years articifl chromosomes) contain telomeres, an origin of replication, and centromere and are extensively used to clone DNA in yeast. With selectable markers (TRPI and URA3) and a cluse of retrscition stes, DNA inserts raning from 100 kb ro 100 kb can be cloned and insertediinto yeast. Since yeasr, being a eukaryote, undergoes many of the typical RNA and protein processing stop of other, more complex eukaryotes, the advantees are numberous when working with eukaryotic genes.

A probe is any DNA or RNA that is complementary to some art of atarget gene or sequence. Probes are used to identify and/or locate a particular nucleic acid sequence among a pool of sequence.

The total number of molecules after 15 cycles would be 16,384, or (2) ^14.

a cDNA library provides DNAs from RNA transcripts and is, therefore, useful in identifying what are likely to be functional DNAs. If one desires an examinatiojn of noncoding as well as coding regiojns, a genomic library would be more useful.

Option (b) fits the expectation because the think band in the offspring probably represents the band at approximatelt the same position in both parents. The likelihood of such a match is expected to be low in the general populatiojn.

(a) Heating to 90=-95 degrees Celsius denatures the double-stranded DNA so that is dissociates into single strands. It usually takes about five minutes, depending on the length and GC content of the DNA (b( lowering the temperature to 50-70 allows the primers to bind to the denatured DNA. (c) Bringing the temperature to 70-75 allows the heat-stable DNA polymerase an opportunity to extend the primers by adding nucleotides to the 3- ends of each growing strand. Each PCR is designed with specific temperatures (not ranges) based on the characteristics of the DNAs (template and primers).

Taq polymersae is from a bacterium called Thermus aquaticus, which typically lives in hot springs. It is heat stable like some other enzymes used in PCR that are isolated form thermal vents in the ocean floor.

ddNTPs are analogs of hthe "normal" deoxyribonucleotide triphosphates (dNTPs), but they lack a 3'-hydroxyl group. As DNA synthesis occurs, the DNA polymerase occationally inserts a ddNTP into a gorwing DNA strand. Since there is no 3'-hydroxyl group, chain elongatiojn cannot take place, and resulting fragemnt are formed, which can be separated by electrophoresis. Where the ddNTP was incorporated then length of each strand, and therefore, the positiojn of the particlar ddNTP are established and sused to eventually provide the base sequence of the DNA.

It is likely that the DNA that served as the template in the sequencing reaction was not pure so that the same position (length), more than one type of ddNTP could be incorporated. This could be due to natural polymorphism, often called single nucleotide polymorphisms (or SNPs) or impure samples.

FISH involves the hybridzaion of a labened probe to a complemnentary strectch of DNA in a chromosome. As such, it can be used to locate a specific DNA sequence (oftena gene or gene fragment) in a chromosome. Spectral karyotyping uses FISH to detect individual chromosomes, a distinct advantage in identifying chromomsl abnormalities.

In general, when DNA fragments of interest have been incorporated into a plasmid the result is a change in the function of a gene or genes in the plasmid. For instance, if one inserts a piece of DNA into the ampicillin resistance gene, following transformation, the bacterium is not longer resistant to ampicillin. Other techniques involving "insertional mutagenesis" might involve using a medium-driven color change in bacteria that contain a recombinant plasmid.

The choice of method often depends on a variety of circumstances. Sometimes probes are used to screen a library. Such probes may be used to identify particular genes in gels or from membranes containing DNA from lysed bacteria. If appropriate primers are available, PCR can be used to identify particular genes of interest.

Purified genomic DNA is first denatured and then annealed to specific primers. Once primers have anneadled, they are extended. The process is repeated to produce many copies of a specific DNA molecule.

Whereas earlier advances relied on single-tube, fluorescent labeling technologies, newer approaches involved solid-phase metholodologies in which beads are attached to DNA fragments and amplified by PCR in water droplets in oil. Coupled with pyrosequencing, processes have been grealty accelerated. New trends often involve nanotechnology and solid support systems. The overall goal is to increase the speed and inaccuracy of seuquencing white reducing the per base cost.