BITC2441 Molecular Biology Techniques
Name __________________
Molecular Diagnostics by Buckingham & Flaws
Guided Lecture Notes
Chapter 6. Analysis and Characterization of Nucleic Acids and Proteins
After reading the whole chapter, answer the following questions into your lecture notebook. Leave spaces for additional
information that might come up during lecture. This preview of the lecture and will allow you to minimize the time needed to
enter lecture notes into your notebook during the lecture. With your notes outlined ahead of time, you will be able to ask
questions during lecture over information that you need help with.
After answering all the questions, sign the question sheet and circle the questions that you want to have emphasized during
lecture, either because you are unsure of your answer or because you need clarification. Hand in this sheet with your circled
questions at the beginning of class. You will not turn in your lecture notebook; you will be given a completion grade for your
work.
HYBRIDIZATION TECHNOLOGIES
1.
What are the targets, probes, and purposes of Southern, northern, western, and southwestern blots?
2.
What are the steps required to perform a Southern blot?
a. What directs the choice in restriction enzyme used?
b. How much gDNA is digested with restriction enzyme?
c. Describe the appearance of a gel after running high quality gDNA digested by a restriction enzyme.
d. Why is it necessary to blot the gel DNA bands to a membrane prior to detection by a hybridization probe?
e. How is the DNA denatured and why is the DNA denatured prior to transfer to a membrane?
f. What types of membranes can be used to blot DNA? Make a table listing their advantages and drawbacks.
g. How are membranes prepared for transfer of DNA?
h. What methods are used to blot DNA? Make a table listing their advantages and drawbacks.
i. How is DNA permanently immobilized to the membrane?
j. How is the membrane prepared for hybridization and why is this step necessary?
3.
Compare the steps required to perform a northern blot with those of a Southern blot.
a. How much total RNA or polyA RNA is loaded on a northern blot gel?
b. Why must denaturant be removed prior to blotting RNA onto a membrane?
4.
What is the basis of protein separation in SDS-PAGE and in IEF gels?
a. What is the denaturant in SDS-PAGE gels?
b. Describe how a 2-dimensional protein gel is performed.
c. How are proteins renatured prior to blotting, and why is this step necessary?
d. What types of membrane are generally used to blot proteins?
e. How is the membrane blocked prior to probing with an antibody, and why is this step necessary?
f. Describe how a western blot is used to test for HIV infection.
PROBES
5.
What forms do probes for Southern and northern blots come in?
6.
What are used for probes of a western blot?
7. Make a list of the ways that DNA probes can be created, indicating which is the more convenient for short probes and
which are necessary for longer probes.
a. What determines the best probe length for an application?
b. How are dsDNA probes prepared prior to hybridization?
8.
What is an advantage of RNA probes?
a. How are RNA probes generally created?
b. What special precautions need to be taken when preparing RNA probes for a northern blot?
c. How can nonspecific binding of RNA to membranes after hybridization to target be eliminated?
9.
What are some advantages of PNA and locked nucleic acid probes?
10. What are used for probes in western blots?
1
a.
b.
c.
How are polyclonal antibodies made?
How are hybridomas made for making monoclonal antibodies?
What are some advantages and drawbacks in the use of monoclonal antibodies?
11. How are nucleic acid probes commonly radiolabeled and what are some drawbacks in this method of probe labelling?
12. What are some nonradioactive adducts used for labeling DNA and RNA probes?
13. Make of list of enzymes that are used to label DNA probes, describing the types of label that is introduced.
14. How are RNA probes labeled?
15. What factors must be considered when designing hybridization probes?
a. Why must high GC content be avoided in hybridization probes?
16. How are secondary antibodies used to detect antibody-binding on a western blot?
HYBRIDIZATION CONDITIONS, STRINGENCY
17. What factors affect stringency of hybridization probes?
18. What factors affect the melting temperature of a hybridization probe?
a. What difference in Tm does a 1% difference in sequence complementarity have?
b. How much higher is the Tm of a DNA:RNA hybrid compared to a DNA:DNA hybrid?
c. How much higher is the Tm of a RNA:RNA hybrid compared to a DNA:DNA hybrid? (Note typo on p. 108!)
d. How does the effective hybridization temperature generally compare with the T m of a hybridization probe?
e. What effect do probe labels have on Tm?
19. A Cot (ssDNA concentration times time to reanneal) curve is Cot plotted against absorbance at 260 nm. Since ssDNA
absorbs more strongly than dsDNA, the reannealing can be followed by monitoring absorbance. What does a C ot curve
reveal about sequence complexity?
20. Why is the volume of hybridization buffer kept to a minimum?
21. Why is formamide generally included in a hybridization buffer using an RNA probe, and what effect does the formamide
have on the optimal temperature of hybridization?
22. How long does hybridization take when using short probes (<20 bases), and when using long probes (>1000 bases)?
a. What can be done to decrease the required hybridization time?
DETECTION SYSTEMS
23. How are bands detected following hybridization with a radiolabelled probe?
24. How are bands detected following hybridization with a digoxygenin or biotin labeled probe?
a. What 2 enzyme systems are generally used to detect labeled target bands?
b. What 2 types of signals are produced in these detection systems?
c. What are the advantages and drawbacks to the use of chemiluminescence, compared to radioisotopic signals?
d. What are the advantages and drawbacks to the use of chromogenic, compared to chemiluminescence signals?
INTERPRETATION OF RESULTS
25. In what situation can a Southern blot detect small delections, additions, or substitutions?
26. What is an internal control, and why is one necessary when using a northern or western blot to estimate levels of gene
expression?
ARRAY-BASED HYBRIDIZATION
2
27. Compare the steps required to perform a dot or slot blot, compared to those of a Southern blot.
a. In what situations are the electrophoresis step not necessary in a blot analysis?
b. What advantages are there to doing slot blots, compared to dot blots?
c. How can a control be designed to rule out cross-hybridization artifacts?
28. What is a reverse dot blot, and what advantage does this technique offer?
29. What are macroarrays immobilized onto?
a. What limits the utility of the routine use of macroarray assays?
30. How many spots of probes (aka “targets”) can be spotted onto a glass slide in microarray technology?
a. Why are gene targets generally printed in triplicate on microarrays?
b. What types of targets are used in printing micrarrays?
c. What types of technologies are used to print microarrays?
d. How are targets produces in gene chips, and what is the capacity of this type of microarray?
e. How are samples prepared for array analysis?
31. How are changes in gene expression determined in expression arrays?
a. What are some reasons that only relative, and not absolute, quantitation is possible with microarrays?
SOLUTION HYBRIDIZATION
32. In what ways are the steps in performing solution hybridization the opposite to the steps in preparing a Southern blot?
33. What enzyme is generally used for solution hybridization assays of RNA?
a. What is the useful information from this type of assay?
34. What does a gel mobility shift assay detect?
KEYWORDS
recognition sites
restriction map
restriction fragment length
polymorphisms (RFLPs)
hybrids
Southern/northern/western/southwestern
Blots
probe
depurination
nylon
nitrocellulose
DEAE cellulose (diethyl amino ethyl)
CM cellulose (carboxymethyl)
PVDF (polyvinyl difluoride)
UV cross-linking
capillary transfer
electrophoretic transfer
vacuum transfer
Denhardt solution
Salmon sperm DNA
SSC
SSPE
2-dimensional electrophoresis
peptide nucleic acids (PNAs)
locked nucleic acids
pGEM plasmid
T7 polymerase
SP6 polymerase
polyclonal/monoclonal antibodies
haptens
hybridomas
biotin
digoxygenin
end-labeling
terminal transferase
T4 polynucleotide kinase
nick translation
DNA polymerase III
random priming
stringency
melting temperature (Tm)
sequence complexity
Cot analysis
dextran sulfate
3
polyethylene glycol
polyacrylic acid
streptavidin
horseradish peroxidase (HRP)
alkaline phosphatase (AP)
chemiluminescent
chromogenic
signal-to-noise ratio
background noise
dot/slot blots
reverse dot blot
macroarrays
microarrays
gene chip
arrayers
photolithography
microelectronic arrays
expression arrays
comparative genome hybridization
reference DNA
solution hybridization
S1 nuclease mapping
gel mobility shift