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Related Concept Videos

Ribosome Profiling02:24

Ribosome Profiling

Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique helps...
RACE - Rapid Amplification of cDNA Ends02:35

RACE - Rapid Amplification of cDNA Ends

Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific primer.
Since the...
RNA-seq03:21

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
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Leaky Scanning02:28

Leaky Scanning

During most eukaryotic translation processes, the small 40S ribosome subunit scans an mRNA from its 5' end until it encounters the first start AUG codon. The large 60S ribosomal subunit then joins the smaller one to initiate protein synthesis. The location of the translation initiation is largely determined by the nucleotides near the start codon as there may be multiple translation initiation sites present on the mRNA.  Marilyn Kozak discovered that the sequence RCCAUGG (where R stands for...
Real Time RT-PCR02:57

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

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Related Experiment Video

Updated: Jul 8, 2026

De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data
08:23

De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data

Published on: February 18, 2022

Complete open reading frame (C-ORF) technique: rapid and efficient method for obtaining complete protein coding

Dong-chul Kang1, Paul B Fisher

  • 1Hallym University, Ilsong Institute of Life Science, Anyang, Kyeonggi-do, Republic of Korea.

Methods in Molecular Biology (Clifton, N.J.)
|January 26, 2008
PubMed
Summary

A new method, complete open reading frame (C-ORF) technique, successfully clones full-length cDNA when standard rapid amplification of cDNA ends (RACE) fails. This cost-effective approach offers broad utility for gene cloning in research laboratories.

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Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames
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Published on: April 11, 2019

Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Standard methods for obtaining full-length cDNA clones via PCR, known as rapid amplification of cDNA ends (RACE), are not always successful.
  • There is a need for improved and reliable techniques to ensure complete cDNA sequence acquisition.

Purpose of the Study:

  • To introduce and validate a novel method, the complete open reading frame (C-ORF) technique, for cloning full-length cDNA.
  • To demonstrate the efficacy of C-ORF in situations where conventional RACE protocols have failed.

Main Methods:

  • The C-ORF technique utilizes a degenerative stem-loop annealing primer to provide the 5' PCR primer site.
  • This primer anneals randomly to first-strand cDNA and promotes second-strand synthesis from the cDNA end.
  • The method employs only reverse transcriptase and Taq polymerase, avoiding additional enzymes.

Main Results:

  • The C-ORF technique has proven successful in cloning full-length cDNA in cases where standard RACE methods were unsuccessful.
  • The method demonstrated applicability across genes with varying GC-rich content, despite a slight GC preference.
  • Successful cloning of both known and unknown genes was achieved using this novel approach.

Conclusions:

  • The complete open reading frame (C-ORF) technique offers a robust alternative for full-length cDNA cloning.
  • Its simplicity, cost-effectiveness, and success in challenging cases make it a valuable tool for molecular biology laboratories.
  • C-ORF expands the toolkit for gene cloning, particularly for difficult-to-clone sequences.