(8/30/10) - Molecular Medicine Techniques



1. List the steps involved in DNA and RNA extraction 

1) Cellular lysis.

2) Removal of RNA or DNA.

3) Removal of proteins and lipid debris.

4) Precipitation of DNA or RNA.

5) Solubilization of the isolated DNA or RNA.

'''2. Describe the quantitation of isolated nucleic acids using ultraviolet absorbance. '''

UV light with a 260 nm wavelength passes through a DNA or RNA solution, and the bases absorb some of the light. For each absorbance unit read by a spectrophotometer at 260 nm, there is about 50 micrograms per milliliter of DNA or 40 micrograms per milliliter of RNA in the solution. You can also measure the protein contamination by comparing the ratio of absorbance at 260 nm to the absorbance of 280 nm. Values greater than 1.8 indicate minimal protein contamination.

'''3. Be aware of the different enzymes used in the manipulation of nucleic acids. Give examples of techniques that employ DNA polymerase, reverse transcriptase and ligase. '''

DNA polymerases allow for the study of DNA in the cell, though lacking exonuclease activities to sequencing itself. Reverse transcriptase is used in cDNA synthesis. Ligases link 5' phosphate and 3' hydroxyl ends via phosphodiester bond creating circular DNA.

'''4. Describe the action of restriction endonucleases on double stranded DNA. Distinguish between enzymes that result in DNA cut with blunt ends and those that result in DNA cut with “sticky” ends. '''

Restriction endonucleases cut double stranded DNA at specific nucleic acid sequences, thus allowing the accurate prediction of the size of the DNA fragments digested by a known restriction enzyme. Those enzymes that leave 'sticky ends' leave a slight overhand in each direction, allowing for the insertion of a genetic sequence into the DNA. SmaI cuts blunt ends, while EcoRI and PstI leave sticky ends.

'''5. Describe the role of nucleic acid probes. List the different types of labels that can be covalently attached to probes. Distinguish direct and indirect probe labeling. '''

Probes are reporter molecules (such as radiation or florescence) attached to a nucleic acid of known sequence. These bond to complimentary sequences of DNA and reveal the presence of these DNA fragments using the reporter molecules. Direct labels are a type of nonradioactive label that consist of fluorescent labels and enzyme reporters that are coupled directly to the DNA. This offers the advantage of rapid analysis. Indirect labels are small affinity labels attached to nucleotides and do not generate detectable signals on their own, but require a high-affinity binding partner that is labeled usually with enzymes or fluorescent labels. The enzyme activity shows the physical presence of the probe.

'''6. Understand the principle behind the separation of nucleic acids by electrophoresis. '''

DNA and RNA are separated out by their molecular weight by being pulled through a gel under an electrical field. Since the nucleic acids are negatively charged, they move towards the positive electrode (the anode). The larger molecules have more difficulty moving through the gel matrix, so they don't move as far.

'''7. List 4 applications of PCR in molecular medicine. '''

Can be used to detect virtually all common genetically inherited diseases when the defective gene locus has been identified. It is used in forensic medicine for the identification of an individual based on minimal amounts of sample DNA. It is used in oncology to detect minor structural changes and arrangements in single copy genes as well as to detect minimal residual disease after therapy. It can be used to detect any pathogen when only a limited DNA or RNA sequence is known.

'''8. Understand the steps involved in DNA amplification by polymerase chain reaction (PCR). Distinguish PCR from RT-PCR '''

1) Denature the DNA.

2) Anneal the primers.

3) Extend the sequence.

4) Rinse and Repeat.

RT-PCR has the additional step at the beginning of using reverse transcriptase to convert RNA to its DNA complement (cDNA).

9. Be able to predict the fold amplification of a target DNA fragment after PCR.

You double the amount of DNA in every PCR cycle, therefore the amount of DNA fragments is 2n where n is the number of PCR cycles you have done.

10. Devise PCR primers to detect a specific sequence of DNA.

RNA primers will have a complementary sequence to the target DNA.

'''11. Describe the mechanism of nucleic acid sequencing by the Sanger method. Understand the rationale behind the use of dideoxynucleotides as chain terminators. '''

Fragments of DNA will be fractioned into four test tubes, each containing the four dNTPs. Each one will also consist of on ddNTP, where there is no 3' hydroxyl. When these ddNTPs are used for synthesis, the polymerase will no longer be able to add additional dNTPs, and synthesis will stop. The newly synthesized DNA strands are then separated by gel electrophoresis and the sequence of the DNA can be determined by the final gel.

'''12. Describe the technical steps involved in Northern and Southern blotting. State the difference between the two techniques. '''

Southern blot is used when the DNA alteration spans a region not easily amplified by PCR. Endonucleases are used to fragment the DNA, which are then separated by agarose electrophoresis. The fragments are then transferred to a nitrocellulose membrane (which immobilizes them) and incubate this membrane with a single stranded probe designed to be complementary to the target sequence. In Northern blotting, the same technique is used, but RNA is used instead of DNA. Also, RNA has defined lengths, so cleavage with restriction enzymes is unnecessary, and RNA has secondary structure that can affect migration on the gel, so electrophoresis must be performed under denaturing conditions.

'''13. Compare and contrast the different techniques to assess gene expression: RT-PCR, Northern blotting and expression microarray. '''

RT-PCR, Northern Blotting, and Microarrays can all use RNA to assess gene expression. Northern blotting, though, is the only one that uses RNA directly, while the other two use reverse transcriptase to get cDNA of the gene to study. RT-PCR and Northern blotting both use gel electrophoresis to study the RNA.

'''14. Predict the restriction fragment length polymorphism (RFLP) pattern for a given sequence variant. '''

'''15. Understand the difference between PCR RFLP and Southern blotting. Be able to select the appropriate technique for a given diagnostic scenario. '''

Southern blotting allows for visualization of RFLPs when the particular segment you are looking at cannot be adequately amplified by PCR. If a fragment contains more than 100 repeats of a tri-nucleotide sequence, for instance, a Southern Blot must be used. PCR RFLP gels will show all the restriction fragments generated from the PCR product, whereas Southern blot will only show the fragments that can be bound by the chosen probe.

'''16. Define recombinant DNA. List the steps involved in molecular cloning.'''

Recombinant DNA is a form of artificial DNA that is created by combining two or more sequences that would not normally occur together. It is possible with the use of restriction endonucleases. Cloning is done by first inserting the DNA fragment into a vector by using endonucleases and DNA ligase. Then the recombinant DNA is reintroduced into bacterial cells, which are then allowed to multiple. The bacteria containing the clone are then isolated.

'''17. Describe the three common features of cloning vectors. Relate them to their role in the cloning process. '''

Cloning vectors all allow for the insertion of DNA into a host organism, such as a bacterium. Plasmids, virus-based vectors, and large insert capacity vectors are all common cloning vectors. Three common features of plasmids are that they all contain a replicator, a selectable marker, and a cloning site that allows the insertion of a foreign gene into the circular DNA.

'''18. Outline the main applications of molecular cloning. '''

Molecular cloning has been useful in sequencing genomes, amplifying a sequence, expression of a foreign gene (such as those used to produce insulin, growth hormone, erythropoietin, coagulation factors, oxitocin, vaccines against hepatitis B, etc), and studying the affects of in vitro mutagenesis.