Part:BBa_K4305003
TFAM_43aa_BamHI_Forward Primer
This primer is used to amplify the TFAM gene (43 aa- 246aa) using human cDNA using PCR.
Literature Review & Experimental Results:
BamHI is a Type II restriction modification system from Bacillus amyloliquefaciensH which recognizes the sequence GGATCC [1]. By recognizing this DNA sequence, it leaves an overhang of GATC which is compatible with many other enzymes. The BamHI-DNA complex is a sequence-specific endonucleases-DNA complex.
There are multiple experiments that have used BamHI sites on primers to overexpress or amplify a certain gene. In one experiment, primers targeting wild-type TFAM gene carrying BamHI (5'-CGAGGATCCACCATGGCGTTTCTCCGAAGC-3') and Notl (5'GTAGCGGCCGCATACACTCCTCAGCACCATA-3') restriction enzyme sequences were used to amplify full-length TFAM cDNA from SAS cells, which encode wild-type TFAM. This was ultimately used to construct the TFAM overexpressing vector [2]. In another experiment, a recombinant product of TFAM-GFP (green fluorescent protein) fusion was amplifed using primers and cloned into a BamHI- and SalI- digest pG-1 vector [3].
A previous experiment was especially similar to our experiment, involving both TFAM protein and also pET28a vector. Mouse TFAM cDNA was amplifed using the PCR primers and cloned into the Ndel and BamHI sites of pET28a. The resulting fusion protein excluded the N-terminal 32 amino acid mitochondrial localization sequence of TFAM, which was replaced with 23 amino acids containing a 6 x His tag and the thrombin cleavage site [4]. A sense primer that contains a BamHI site has also been used for amplification. In this specific experiment, the DNA fragments for human TFAM and the HA tag were digested with appropriate restriction enzymes and inserted between the BamHI and NheI sites of vector pTRE2hyg [5].
Experimentally, this forward primer was used to amplify the TFAM gene (43aa-246aa) using human cDNA using PCR. During this process TFAM cDNAs from different cell origins, A172 (brain), MCF7 (breast), and MKN45 (stomach), and A549 (lung) were tested via PCR and put into an agarose gel to test which cell has the gene to produce TFAM protein the most. The TFAM-204 mRNA coding sequence (CDS) was multiplied via PCR. The solution included distilled water, forward primer, reverse primer, TFAM-204 cDNA, the mixture of optimized DNA polymerase, dNTP, and the buffer. For an hour and a half, denaturation, annealing, and elongation were repeated to amplify TFAM-204 cDNA. After PCR, the solutions were put into an agarose gel, where the protein was expressed.
The results of the agarose gel showed that human lung cDNA expressed the most TFAM-204 protein, indicated by the width of the band with 636 base pairs; thus, human lung cells were used throughout the remainder of the experiment. The results also implied that all the TFAM cDNA was amplified without errors.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 4
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References:
[1] Brooks, J.E et al. Cloning the BamHI restriction modification system. Nucleic Acids REs. 17, 979-997 (1989).
[2] Hsieh, Yi-Ta et al. Mitochondrial genome and its regulator TFAM modulates head and neck tumourigenesis through intracellular metabolic reprogramming and activation of oncogenic effectors. Cell Death & Disease. 12, 961 (2021).
[3] Schena, M et al. Vectors for constitutive and inducible gene expression in yeast. Methods Enzymol. 194, 389-98 (1991).
[4] Brown, Timothy A. et al. Mitochondrial Transcription Factor A (TFAM) Binds to RNA Containing 4-Way Junctions and Mitochondrial tRNA. Plos One (2016).
[5] Kanki, Tomotake et al. Architectural Role of Mitochondrial Transcription Factor A in Maintenance of Human Mitochondrial DNA. Molecular and Cellular Biology (2004).
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