Advanced Molecular Techniques (Bio. 205): a Course Overview.
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Advanced Molecular Techniques (Bio. 205): a Course Overview. Fall 2004 Bio. 205 students and Dr. Steven White — Department of Biology, San Jose State University, San Jose CA 19192. 1 2 3 4 5 6 7. Introduction

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Advanced Molecular Techniques (Bio. 205): a Course Overview.

Fall 2004 Bio. 205 students and Dr. Steven White — Department of Biology, San Jose State University, San Jose CA 19192

1 2 3 4 5 6 7

Introduction

Students in molecular biology must gain practical experience in a variety of modern laboratory techniques if they are to have any real understanding of how experimental designs are developed and carried out, if they are to make effective use of their time in the research lab, if they are to be competitive in today’s job market etc. Bio. 205 was developed to address some of that need for practical experience, with experiments and techniques presented in a logical progression and linked format (with one experimental result used as input to another) to more closely approximate industrial or graduate “thesis research” experience. The technique summaries presented here are meant to provide the reader with an “experimental context” (there is simply insufficient room to include actual methodological detail), but all data is “student group generated” and representative of all group results. [Note that parallel group research projects were also performed “off hours” as an additional course requirement for Bio.205, but results there will be the subject of another poster.]

Fig. 14. Example of in vitro transcription reaction products run out on a 1% native agarose gel just prior to use with Ambion’s photoactivatable psloren- biotinylation system. The biotinylated probe was then used in a Northern.

Note that the robust production of transcript in lane 3 is due to the use of Ambion’s MEGAscript in vitro transcription system, which is optimized for use with template designed to produce transcripts of < about 350 bases. This result suggests that short RNAs are more efficiently synthesized using this modified transcription system.

10 1 0.1 0.01

Fig. 7. Example of Southern hybridization results. The biotinylated Hind III Lambda fragment was used to probe dilutions of a Lambda DNA digest run in a background of calf thymus DNA, which allowed highly quantitative analysis of the sensitivity of this method.

procedure using Novagen Inc reagents) linearized, blunt-ended pST Blue-1 supplied by Novagen. Following ligation, the products were employed in a chemical transformation according to protocols supplied by Promega or Novagen, respectively. Transformants were plated on LA/Amp/IPTG/XGal plates for blue/white screening, and numerous white colonies subsequently grown up for plasmid isolation. The plasmids so derived were screened by PCR using T7 and SP6 priimers to identify those with appropriately sized inserts.

Cycle (DNA) Sequencing: Recombinant plasmids resulting from the TA and blunt-end cloning experiments were then employed as templates in either a “Big Dye” or “d-Rhodamine” cycle sequencing protocol. The chain extension products were precipitated, washed, lypholyzed and resuspended following standard protocols (see ABI Prism 310 sequencing manual) prior to capillary electrophoresis (1, 2).

cDNA production for cDNA library construction: Mouse mRNA was used in first and second strand cDNA synthesis using Novagen’s OrientExpress cDNA synthesis kit following the manufacturers protocols (1, 2).

Construction of a cDNA library in Lambda Phage: The resulting double stranded cDNAs were end-polished, methylated in vitro, ligated to linkers, restriction digested and and the linker digestion products and free linkers removed using molecular filtration spin columns. The “ligation-ready” cDNAs were then ligated to Novagen’s LambdaScreen pre-digested Lambda arms and then packed “in vitro” using Novagen’s packaging reagent system. Packaged recombinant Lambda phage were then used to infect ER1647 E. coli host cells, plated in top agar over agarose using standard methods (Maniatis et al), and the resulting viral plaques counted and ultimately harvested (using the “flood plate” technique) and stored at -70 oC for use by subsequent classes (1, 2).

[Lane 1= 100bp ladder, Lane 5= Low mass InVitrogen DNA ladder. Other lanes are various student group results.

(A)

(B)

(C)

1 2 3 4 5 6 7

Fig.15. Examples of group Northern blots. A typical denaturing agarose gel run (A) and the corresponding X-ray film following hybridization, washing and detection (B). Film in (C) shows another blot. Numbers below A indicate amount of total RNA (lanes1-6) or mRNA (lane 7) loaded in each lane.

Numbers above each lane correspond to total amount of Lambda DNA loaded (in nanograms), while numbers below are target DNA mass detected (in picograms).

470 47 4.7 0.47

Fig. 8. Results of 2-dimensional protein gel electrophoresis using two different protein extraction protocols (IEF gel pH gradient of pH 4-7, SDS-PAGE gradient of 8-16% polyacrylamide). Many proteins isolated by the second protocol clearly are underrepresented.

Mouse Tissue

Linearized transcription vector containing insert

Total RNA isolation

Overview of Materials and methods

All reagents were prepared by staff, obtained commercially or donated. We especially acknowledge both the Ambion and Novagen companies for their generous support.

Plasmid Isolation and Characterization: Plasmids were isolated using both “in house” prepared reagents for a standard alkaline lysis protocol (1, 2) and commerically obtained Qiagen SpinPreps (Qiagen Inc.) to allow comparison of average yield, degree of contamination (as measured by OD260/280 ratio, 210nm-300nm absorption spectrum analysis and agarose gel electrophoresis [AgGE]) and functionality (as measured by capacity for restriction digestion and religation). Parallel DNA samples were “spiked” with contaminants (ie chaotropes, solvents, protein, salts etc) prior to spectrophotometry, AgGE or functional tests for purposes of comparison. Additionally, DNAs were sometimes further purified and concentrated using spin ultrafiltration via Amicon-30 spin filters (Amicon Inc).

Restriction Digests & Agarose Gel Electrophoresis (AgGE): All restrictions were performed according to standard protocols (1, 2), using reagent grade water and commercially obtained enzymes and 10X buffers (Promega Inc). Native AgGE runs (using TAE, TBE or MOPS buffers) were performed using “in house” prepared reagents (1, 2). Denaturing (formamide/formaldehyde& MOPS) AgGE runs for RNA utilized reagents supplied by Ambion Inc, and were performed using the Ambion’s protocols (essentially those described in references 1 and 2). DNA was isolated from excised agarose gel bands using a QIAquick kit (Qiagen Inc) based protocol.

Genomic DNA isolation and PCR: E.coli genomic DNA was isolated using both “in house” prepared reagents and a standard CTAB protocol (2) and via a Gentra DNA isolation kit. The DNAs were analyzed by AgGE, then used in a PCR reaction (3) to amplify a 730 bp fragment of the Trp A gene. PCR product was then analyzed by AgGE against mass and size standards. [Originally we intended to run an second Southern using this TrpA gene fragment as a probe, but scheduling problems prevented it.]

DNA labeling for use in Southern Hybridization: Probe DNA (a 2.3 kbp Hiind III fragment from Lambda phage) was labeled using the Phototope system kit (New England Biolabs Inc) via a random priming protocol (1, 2) using both biotinylated primers and a biotinylated dNTP.

Southern Hybridization: Varying amounts of Hind III digested Lambda genomic DNA (target DNA) diluted in irrelevant calf thymus DNA (to add sequence complexity to the target population) were electrophoresed on a native agarose gel and transferred to nylon via capillary blotting according to standard protocols (Maniatis et al). Hybridization (probe at 20 ng/ml) was carried out overnight at 68 oC following the NEB protocol with “standard” hyb solution or 42 oC with “Ultrahyb” solution, and hybrids detected using the NEB Phototope Detection system following the NEB protocol. X-ray films were developed using Kodak GBX developer & fixer solutions following standard Kodak protocols (1, 2, 5).

Protein isolation from E. coli using various extraction protocols. Bacterial proteins were isolated using one of 3 different extraction protocols in which the nature and/or concentration of the non-ionic detergent were varied. Protein content of each fraction was then determined using BioRad’s RC DC protein assay kit.

2D-PAGE (IEF and SDS-PAGE) of extracted bacterial proteins. Samples were diluted in BioRad’s IEF sample loading buffer and applied to pH 4-7 Immobiline IEF strips and subjected to isoelectric focusing on a BioRad Protean IEF unit following their recommended protocol. The focused IEF strips were then soaked in SDS-PAGE sample buffer and applied to a Criterion 10.5 to 14% gradient SDS-PAGE gel, electrophoresed, and stained using Ruby Red following manufacturers protocols (6).

Isolation of Mouse Total RNA: Total RNA was isolated from mouse liver and heart tissue using both the TRI reagent (Sigma Inc) and TotallyRNA prep (Ambion Inc) protocols following manufacturer’s instructions (1, 2, 4). The RNAs were analyzed for yield, purity, size range and degradation via spectrophotometry and AgGE. Total RNAs were then stored in DEPC-treated water at -70 oC.

Isolation of Mouse mRNA: mRNAs were isolated from total RNA via Ambion’s Poly(A) Purist kit and protocol (4). The resulting mRNA preps were then analyzed via both spectrphotometry and AgGE.

In Vitro Transcription: A linearized plasmid containing a 285 bp fragment of the mouse actin gene was used in a “run off” in vitro transcription protocol utilizing either Ambion’s MAXIscript or MEGAscript In Vitro RNA synthesis kit. The transcripts were then analyzed by AgGE, and purified for RNA labeling (1, 2, 4).

RNA labeling for use in Northern Hybridization: RNAs to be employed as hybridization probes were post-synthetically biotinylated using Ambion’s BrightStar Psoralen-Biotin labeling system.

Northern Hybridization: Mouse total and mRNA samples were run out on denaturing gels and blotted by capillary transferto nylon membranes following standard protocols (Maniatis et al). Biotinylated probe (appeox. 0.1 ng/ml) was diluted in Ambion’s Ultrahyb solution, hybridized at 42 oC overnight, and the blot then washed and hybrids detected using Ambion’s NorthernMax and BrightStar Detection systems (1, 2, 4, 5).

RT-PCR: Mouse total RNA and class-designed primer sets were employed to specifically amplify a mouse Nkx2.5 gene fragment or actin gene fragment using Qiagen’s “One-Step” RT-PCR system, with the products subsequent used in both blunt end and TA cloning efforts. Following RT-PCR, the products were analyzed by AgGE, then cleaned or band purified prior to their use in ligation (1, 2, 3).

“TA” and Blunt end Cloning: The purified DNA fragments generated by RT-PCR were next ligated into both a linearized “T”-ended vector (pGEM T-Easy from Promega Inc) or (following an “end polishing”

Fig. 2. Flow chart summarizing experimental design for the second portion of the course.

In Vitro Transcription and cleanup

mRNA isolation

RT-PCR and cleanup

Denaturing AgGE and Capillary Blot

AgGE and band isolation

Riboprobe Labeling

TA and Blunt end Cloning

18 9 4.5 2.25 1.12 0.62 1

Plasmid isolation

Northern Hybridization and Detection

cDNA Library construction in Lambda Phage

Cycle DNA Sequencing

Fig. 16. Photographs at right show representative plates of recombinant Lambda phage, as viral plaques on a lawn of host E. coli. Mouse mRNA was used to create cDNA libraries cloned into Novagen’s LambdaScreen vector.

Results

Fig. 3. Example of (A) absorbance scans of pure DNA vs (B) DNA in the presence of a contaminant (in this case, 5% v/v guanidine hydrochloride), (C) “spot gel” showing DNA dilution series, and (D) “short run” gel showing DNA dilution series (both used to quickly estimate DNA concentrations in small volumes of unknowns).

1 2 3 4 5 6 7

Fig. 9. Examples of mouse total RNA isolated using Ambion RNAeasy (lanes2-4) vs total RNA isolated using TRI reagent (lanes 5-7). In both cases the RNA appears intact (sharp 28s and 18s bands). Note, however, that the RNA isolated using TRI reagent was often significantly contaminated with genomic DNA, and thus frequently required extensive DNase digestion prior to use in RT-PCR or cDNA library construction. [Gel on right is RNAeasy prep only]

Conclusions

Results shown here provide some indication of the technical experience gained by the Bio205 students. Other experience, which is difficult to present in this condensed format but is nonetheless important, involved their skills development in overall project design, individual experimental design, technique problem diagnosis and treatment, database mining, literature analysis and goals assessment. In addition to the accompanying instructor lectures (in which underlying theory and technical aspects are discussed), students take two 3 hour written exams, carry out a supplementary research project (beyond the work presented here), create and turn in (for graded evaluation) a research notebook detailing all their work in the course and discussing their results, and give a 15 min Powerpoint presentation (ASM or ASCB format) on one of various instructor-designated research topics. Despite the rigor of the course, students unanimously agreed the experience was extremely valuable.

(A)

(B)

(D)

(C)

Note that the OD260/280 ratios in A and B are essentially identical (1.75 vs 1.69) while their absorption spectra clearly are not, illustrating the importance of spectrum analysis in assessment of DNA purity.

Fig. 4. Examples of 1% agarose TAE gel runs. (A) Dilutions of purified plasmid (uncut). (B) Plasmid restricted with Hind III to liberate insert. (C) Gel following insert band incision. (D) Gel showing purified inserts ready for biotinylation.

Fig. 11. Example of RT-PCR products generated using total RNA and a thermal gradient cycler to identify the optimal primer annealing temperature for amplifying a mouse Nkx2.5 gene fragment. Products anlyzed on a 2% native agarose gel.

1 2 3 4 5 6

Fig. 10. Example of purified mRNA (isolated using Ambion’s MicroPolyA Pure kit, starting from total RNA), prior to use in Northern blot experiments and cDNA library construction.

Bacterial cells

Plasmid isolation and purity analysis

Genomic DNA isolation

Protein extraction and sample prepartion

Fig. 1. Flow chart summarizing experimental design for the first portion of the course.

(B)

Restriction digestion and AgGE

(C)

(D)

(A)

Isoelectric focusing

Band purification

PCR (at various annealing temps and/or template amounts)

Random priming-based DNA labeling and probe purification

SDS-PAGE (2-Dimensional gel electrophoresis)

Literature cited

1. Sambrook, J., Fritsch, E.F., and T. Maniatis. 1989. Molecular Cloning, A Laboratory Manual. Cold Springs Harbor Press.

2. Ausubel, F. et al. 1997. Short Protocols in Molecular Biology. John Wiley & Sons.

3. Newton, C.R., and A. Graham. 2000. PCR (Second Edition). Springer Pub. Co.

4. Farrell, R. 1993. RNA Methodologies: A Laboratory Guide for Isolation and Characterization. Academic Press.

5. Hames, B.D., and S.J. Higgins. 1985. Nucleic Acid Hybridization: A Practical Approach. IRL Press.

6. Hames, B.D., and D. Rickwood. 1990. Gel Electrophoresis of Proteins: A Practical Approach. IRL Press.

Southern blot, hybridization and detection

AgGE

Fig. 12. Example of RT-PCR products generated using mouse total mouse RNA and actin primers.

[Optimal annealing temp = 60 oC, lane 6]

Fig.6. Examples of preliminary Southern hybridization experiments using (A) standard hyb buffer (Maniatis et al) vs (B) Ambion Ultrahyb buffer.

Fig. 5. Example of 2% agarose gel run of TrpA PCR product. This fragment will be cloned for use in a later Bio205 class.

(A)

Acknowledgments

We thank D. Leeve and L. Goff for technical assistance, and all those at both Ambion and Novagen Corporations for their generous support.

Fig. 13. Examples of blunt end cloning vectors (A) and “TA” cloning vectors (B) used in the construction of recombinant plasmids containing mouse Nkx2.5 or mouse actin RT-PCR fragments. Both fragments were successfully cloned in both vectors. Positive actin clones, identified using a T7/SP6 primer-based PCR technique, are shown in (C) and (D).

(A)

(B)

For further information

It is important to note that Bio. 205 is not atypical, but is in fact representative of upper division and graduate level lab courses in the Dept. of Biology as SJSU. See, for example Bio. 205T, Bio. 233, Bio. 234, Bio. 227, Bio. 135/135L, Bio. 124/125, Micro. 141/141L etc. etc. Please visit the SJSU web site, call the SJSU Dept. of Biology or contact Dr. White at [email protected] for more details.

(B)

The Ultrahyb buffer gave a much stronger signal for all groups.

(C)

(D)


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