Lecture 15 Molecular Genetic Analysis and Biotechnology Flashcards
(43 cards)
Southern blot =
Northern blot =
Western blot =
DNA
RNA
Protein
Probe:
DNA or RNA with a base sequence complementary to a sequence in the gene of interest
Gene cloning:
Amplifying a specific piece of DNA via a bacteria cell
Cloning vector:
a replicating DNA molecule attached with a foreign DNA fragment to be introduced into a cell
What are some of the effects and practical applications of molecular genetic analysis?
Molecular genetics has had profound effects on all fields of biology. Whole genomes have been sequenced, structures of genes elucidated, the patterns of molecular evolution studied. Recombinant DNA technology is now used to diagnose and screen for genetic diseases, and gene therapy is being explored. Recombinant DNA is used to make pharmaceutical products, such as recombinant insulin and clotting factors. Genetically modified organisms is changing the lives of farmers and improving agricultural productivity and the quality of food and fiber.
What feature is commonly seen in the sequences recognized by type II restriction enzymes?
The recognition sequences are palindromic, and 4-8 base pairs long
What are restriction enzymes?
Enzymes that recognize and cut DNA at specific nucleotide seqences
What normal role do restriction enzymes play in bacteria?
Restriction enzymes cut foreign DNA, such as viral DNA, into fragments.
How do bacteria protect their own DNA from the action of restriction enzymes?
They modify bases, usually by methylation, at the recognition sites
What are cohesive ends?
Fragments with short, single-stranded overhanging ends
What are blunt ends?
Even-length ends from both single strands
What ends are harder to rejoin?
blunt ends
Which ends are sticky?
cohesive ends
How is gel electrophoresis used to separate DNA fragments of different lengths?
Gel electrophoresis uses an electric field to drive DNA molecules through a gel that acts as a molecular sieve. The gel is an aqueous matrix of agarose or polyacrylamide. DNA molecules are loaded into a slot or well at one end of the gel. When an electric field is applied, the negatively charged DNA molecules migrate toward the positive electrode. Shorter DNA molecules are less hindered by the agarose or polyacrylamide matrix and migrate faster than longer DNA molecules, which must wind their way around obstacles and through the pores in the gel matrix.
After DNA fragments have been separated by gel electrophoresis, how can they be visualized?
DNA molecules can be visualized by staining with a fluorescent dye, such as ethidium bromide, that intercalates between the stacked bases of the DNA double helix, and the dye–DNA complex fluoresces when irradiated with an ultraviolet light source. Alternatively, they can be visualized by attaching radioactive or chemical labels to the DNA before it is placed in the gel.
What can gel electrophoresis not determine?
Nothing about the sequences, but only the size of the fragment
What is the purpose of southern blotting?
Southern blotting is used to detect and visualize specific DNA fragments that have a sequence complementary to a labeled DNA probe.
How is Southern blotting carried out?
DNA is first cleaved into fragments with restriction endonucleases. The fragments are separated by size via gel electrophoresis. These fragments are then denatured and transferred by blotting onto the surface of a membrane filter. The membrane filter now has single-stranded DNA fragments bound to its surface, separated by size as in the gel. The filter is then incubated with a solution containing a denatured, labeled probe DNA. The probe DNA hybridizes to its complementary DNA on the filter. After washing away excess unbound probes, the labeled probe hybridized to the DNA on the filter can be detected using the appropriate methods to visualize the label. For radioactively labeled probes, the bound probe is detected by exposure to X- ray film. Other probe labeling methods detect bound probe using enzymatic reactions that generate luminescence or color.
Give three important characteristics of cloning vectors
(1) An origin of DNA replication so they can be maintained in a cell
(2) A gene, such as antibiotic resistance, to select for cells that carry the vector
(3) A unique restriction site or series of sites to where a foreign DNA molecule may be inserted
Briefly describe two different methods for inserting foreign DNA into plasmids
Restriction cloning: Vector and foreign DNA are cut with the same restriction enzyme, then ligated together with DNA ligase.
PCR fragment cloning: DNA fragments generated by PCR may be ligated to plasmid vectors in either of two ways. One way is to synthesize PCR primers that have restriction sites at or near their 5′ ends. The resulting PCR fragments can be digested with the appropriate restriction enzymes to generate sticky ends for restriction cloning as described above.
Briefly explain how an antibiotic-resistance gene and the lacZ gene can be used to determine which cells contain a particular plasmid
Many plasmids designed as cloning vectors carry a gene for antibiotic resistance and the lacZ gene. The lacZ gene on the plasmid has been engineered to contain multiple unique restriction sites. Foreign DNAs are inserted into one of the unique restriction sites in the lacZ gene of plasmids and the plasmids are transformed into E. coli cells lacking a functional lacZ gene. Transformed cells are plated on a medium containing the appropriate antibiotic to select for cells that carry the plasmid. The medium also contains an inducer for the lac operon, so the cells express the lacZ gene, and X-gal, a substrate for beta-galactosidase that will turn blue when cleaved by β-galactosidase. The colonies that carry plasmid without foreign DNA inserts will have intact lacZ genes, make functional β-galactosidase, cleave X-gal, and turn blue. Colonies that carry plasmid with foreign DNA inserts will not make functional β-galactosidase because the lacZ gene is disrupted by the foreign DNA insert. They will remain white. Thus, cells carrying plasmids with inserts will form white colonies. This is known as blue/white selection.
Briefly explain how the polymerase chain reaction is used to amplify a specific DNA sequence
First, the double-stranded template DNA is denatured by high temperature. Then, primers corresponding to the ends of the DNA sequence to be amplified are annealed to the single-stranded DNA template strands. These primers are extended by a thermostable DNA polymerase so that the target DNA sequence is duplicated. These steps are repeated 30 times or more. Each cycle of denaturation, primer annealing, and extension results in doubling the number of copies of the target sequence between the primers.
What are some of the limitations of PCR?
PCR amplification is limited by several factors. One is that sequence of the gene to be amplified must be known, at least at the ends of the region to be amplified, in order to synthesize the PCR primers. Another is that the extreme sensitivity of the technique renders it susceptible to contamination. A third limitation is that the most common thermostable DNA polymerase used for PCR, Taq DNA polymerase, has a relatively high error rate. A fourth limitation is that PCR amplification is usually limited to DNA fragments of up to a few thousand base pairs; optimized DNA polymerase mixtures and reaction conditions extend the amplifiable length to around 20 kb.
What is real-time PCR?
Real-time PCR uses fluorescent probes to detect the formation of specific PCR products, and a sensitive instrument to quantify the amount of PCR product formed after each cycle of PCR, while the reaction is proceeding. This technique is used to quantify absolute or relative amounts of template DNA in samples.
