Students participated in hands-on research, in addition to lecture, to improve their understanding of molecular biology. Hands-on research included cloning and sequencing a gene that codes for Glyceraldehyde -3-phosphate dehydrogenase
(GAPDH) in multiple species of plants.
The impact of hands-on learning in students’ understanding of molecular biology was qualitatively assessed. Results confirm that this approach can be effective in increasing students’ confidence with basic laboratory techniques and knowledge of bioinformatics.
The purpose of this research was to provide undergraduate students with the opportunity to learn molecular biology through hands-on experimentation, in addition to traditional lecture and to qualitatively assess how hands-on learning improved their understanding of molecular biology.
During the semester, approximately half of the time in the classroom was spent cloning the GAPC gene in multiple plants. The following semester was spent performing bioinformatics analysis on sequences of GAPC clones. During their hands-on research students accomplished the following:
•   Extraction and purification of genomic DNA from plants
•   Amplification of target DNA using polymerase chain reaction (PCR)
•   Assessment of PCR amplification using gel electrophoresis
•   Purification of PCR products using size exclusion chromatography
•   Ligation of PCR products into a plasmid vector
•   Cloning target DNA by transformation via bacteria
•   Identification and isolation of transformed bacteria using a selection marker
•   Extraction and purification of cloned DNA plasmid from bacteria
•   Sequencing and bioinformatics analysis of cloned genes
•   Troubleshooting for unexpected results and subsequent reassessment
The Cloning and Sequencing Explorer Series kit from Bio-Rad (#166-5000EDU) was used for extraction, cloning, and amplification of the GAPC gene in all plants used. Cloned GAPC fragments were analyzed using NCBI’s basic local alignment search tool, CAP3 Sequence Assembly Program, and the European Bioinformatics
Institute’s sequence translation tool. The plants shown below were used in addition to a pGAP control plasmid containing the GAPC target region from
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Figure 1. Chromosomal locations of the seven GAPDH genes in
The GAPDH gene that was cloned is indicated in the red box (GAPC).
Figure 2. Sequence homology between GAPC in successfully cloned plants compared to chromosomal locations of GAPC in
Exons are shown in red. The portion of
GAPC including the active site of GAPDH was cloned in all five of these plant species.
Figure 3. Multiple alignment of amino acid residues of
GAPDH in successfully cloned plant species compared to coding sequences for cytosolic GAPDH in
(MultAlin).
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1.
What is bioinformatics?
2.
What are the characteristics of a high quality base?
3.
What is NCBI?
4.
What is an “accession number” in NCBI?
5.
What is an E value?
6.
Why may sequencing reads from different sequencing primers of the same miniprep clone be homologous to different GAPC genes?
7.
Does an E value of zero mean that your sequence matched the subject sequence well or poorly?
8.
What is a read? How is it different from a sequence?
9.
What kinds of sequences will be found in a genomic sequence?
10.
What kinds of sequences will be found in mRNA?
11.
Blastx translates a nucleotide sequence in six reading frames and uses each one to query a protein database. Why are there six possible reading frames?
12.
In the Blastx results the letters are no longer limited to A,G,C or T. What do letters in the Blastx results represent?
13.
What is the function of the GAPDH gene?
14.
Why was nested PCR used in this experiment?
15.
What is the structure of a gene in eukaryotes?
16.
What is nested PCR?
17.
What are degenerate primers and why would you use them?
18.
Why did we purify PCR products? Why is that important?
19.
What was the purpose of using Ampicillin in the media used to grow bacteria?
Understanding  of  molecular  biology
Understanding  of  basic  lab  techniques
Ability  to  participate  in  scienti=ic   research
Ability  to  troubleshoot  and  continue   research  when  actual  results  differ   from  expected  results
Understanding  of  PCR
Ability  to  clone  a  gene  using  nested
PCR
Understanding  of  how  to  extract   genomic  DNA  from  plant  cells
Understanding  of  gel  electrophoresis
Ability  to  run  a  gel  using  gel   electrophoresis
Understanding  of  how  to  transform   bacteria  with  a  gene  of  interest
Familiarity  with  DNA  sequencing  and   bioinformatics
Prior  to  cloning
GAPDH
2.2
2.6
2
1.8
2.8
1
1.6
2.8
2.2
1.2
1.4
After  cloning
GAPDH
4.2
4.6
4.2
4
5
4.2
4.6
4.8
5
4.8
4.2
Percentage
Increase
40%
40%
44%
44%
44%
64%
60%
40%
56%
72%
56%
Table 1. Students rated the concepts above on a 1 through 5 scale. Ratings prior to and after cloning exercise are shown, along with the overall percent increase.
1 = poor, 2 = fair, 3 = good, 4 = very good, 5 = exceptional
•   GAPC genes were successfully cloned in five of the six plant species (
) and coding sequences will be published in NCBI’s GenBank.
•   GAPC clones showed high sequence homology when compared to GAPC in
•   Introduction of cloning and sequencing exercises into the teaching of molecular biology was an effective way to increase student learning and retention, as shown in Table 1 and formal classroom assessments.
•   Students reported at least a two-fold increase, on a
1-5 scale, in their understanding of concepts and techniques of molecular biology after completing hands-on exercises of cloning and sequencing.
•   An overall increase of 30.36% was reported in students’ understanding of molecular biology concepts and techniques following hands-on exercises.