Part:BBa_K4305004
TFAM_43aa_XhoI_Reverse Primer
This primer is used to amplify the TFAM gene (43 aa- 246aa) using human cDNA using PCR.
Literature Review & Experimental Results:
In order to clone human TFAM, XhoI sites have been previously used. Restriction sites were introduced by the forward and reverse (CCATCGATC CATTGTGAACACATCTC) primers. The TFAM insert in this experiment was subcloned by the XBaI and XhI sites to yield pBS-TFAM used for in vitro transcription / translation analyses. A tuncated version of TFAM was produced lacking 25 amino acids at the C-terminal end, and the PCR product was cloned into the XbaI and XhoI sites using pBS-TFAM as a template and appropriate primers containing XbaI and XhoI restriction sites [1].
In other experiments, a similar process was repeated. Certain genes have been cloned into different expression vectors using XhoI restriction sites along with others such as NcoI and further expressed and purified specific proteins [2]. XhoI has also been used to digest fragments that are made. For example, an RBS-lambda phosphatase fragment was generated with primers and digested with KpnI and XhoI. This fragment was also ligated into the cloning vector which was previously linearized with BamHI and XhoI [3].
Polymerase Chain Reaction (PCR) is based on three steps required for any DNA synthesis reaction: (1) denaturation of the template into single strands; (2) annealing of primers to each original strand for new strand synthesis; and (3) extension of the new DNA strands from the primers [4]. The XhoI reverse primer has been used in the second step, for annealing primers in order for genes to be synthesized based on the template strand.
Experimentally, the XhoI reverse primer was involved in the PCR reaction. The TFAM cDNAs from different cell origins, A172 (brain), MCF7 (breast), 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 the 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 experiment. The results also implied that all the TFAM cDNA was amplified without errors.
Furthermore, the pET28a vector and TFAM cDNA were then digested by BamHI and XhoI. TFAM cDNA was inserted into the pET28 vector, and the pET28-TFAM vector was transformed into BL21 (DE3) E.coli strain.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 4
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References:
[1] Garstka, Heike L. et al. Import of mitochondrial transcription factor A (TFAM) into rat liver mitochondria stimulates transcription of mitochondrial DNA. Nucleic Acids Res. 31, 5039-5047 (2003).
[2] Litonin, Dmitry et al. Human Mitochondrial Transcription Revisited: ONLY TFAM AND TFB2M ARE REQUIRED FOR TRANSCRIPTION OF THE MITOCHONDRIAL GENES IN VITRO. Jounal of Biological Chemistry. 285, 18129-18133 (2010).
[3] Burger, Michael et al. The TFAMoplex—Conversion of the Mitochondrial Transcription Factor A into a DNA Transfection Agent. Advanced Science. 9, 2104987 (2022).
[4] Delidow, B.C. et al. Polymerase chain reaction : basic protocols. Methods Mol Biol. 15, 1-29 (1993).
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