Genome ORF Analysis and Prediction

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Questions and Answers

What is the first step after sequencing and assembling the genome?

Locate all the protein-coding genes within the genome.

What does annotation mean in the context of genome sequences?

Adding specific structural or functional information about specific regions of sequences or genomes.

How many possible reading frames are there for DNA?

Six possible reading frames.

What is an open reading frame (ORF)?

The continuous nucleotide sequence that begins with a start codon and ends with a stop codon. Signup and view all the answers

What is the difference between a coding sequence (CDS) and an open reading frame (ORF)?

CDS is the real part of the gene translating into a protein; ORF is the segment of DNA between the start codon and stop codon. Signup and view all the answers

All ORFs are coding sequences.

False Signup and view all the answers

What effect does a frameshift mutation have on a gene?

It causes the codons to be read incorrectly, potentially resulting in different amino acids being coded. Signup and view all the answers

What tool is used to find all open reading frames in a given sequence?

ORFfinder. Signup and view all the answers

Match each question about ORFs with the correct description:

Q1 = Find the number of ORFs for this accession code. Q2 = Identify which strand and frame the ORFs are present on. Q3 = Determine the number of nucleotides and amino acids present in these ORFs. Q4 = Using the blast search tool, find the putative function of these ORFs. Signup and view all the answers

What indicates the start of an open reading frame (ORF)?

A start codon Signup and view all the answers

Which of the following is true regarding the length of open reading frames?

Long ORFs help in predicting potential protein-coding regions. Signup and view all the answers

What is the possible outcome if a stop codon interrupts an ORF?

The ORF is considered non-functional. Signup and view all the answers

How many reading frames are available for RNA sequences?

Three Signup and view all the answers

Which of the following describes the nature of codons?

Codons consist of three nucleotides. Signup and view all the answers

What is the primary function of a coding sequence (CDS)?

To provide a protein-coding recipe Signup and view all the answers

Which statement about open reading frames (ORFs) is true?

An ORF must be contiguous between a start and stop codon Signup and view all the answers

What characterizes a frameshift mutation?

Incorrect reading of codons affecting the downstream sequence Signup and view all the answers

In which situation can an open reading frame (ORF) still result in a functional protein despite a mutation?

If multiple codons correspond to the same amino acid Signup and view all the answers

Which information must be known to analyze open reading frames using ORFfinder?

The accession number of the DNA sequence Signup and view all the answers

Study Notes

Introduction

ORF analysis is used to locate protein-coding genes within a genome.
It helps understand the functional aspects of a genome.
Computational gene prediction aims for near 100% accuracy and reduces experimental work.
Annotation adds structural or functional information to sequences or genomes.

Reading Frames

Reading frame is a way of reading a nucleotide sequence.
DNA is read as a sequence of triplets called codons.
In silico gene prediction involves determining the correct ORF.
ORFs begin with a start codon (ATG) and end with a stop codon (TAG, TAA, TGA).
A DNA sequence has six possible reading frames: three on the forward strand (plus strand), three on the reverse strand (minus strand).

Open Reading Frame (ORF)

ORF is a continuous nucleotide sequence.
It starts with a start codon, followed by codons, and ends with a stop codon.
ORFs are potential coding sequences for genes.
ORFs can be translated into proteins.
Functional Clues: Long ORFs indicate protein-coding regions but do not guarantee translation.

CDS (Coding Sequence) and ORF (Open Reading Frame)

CDS is the real part of a gene that is translated into a protein.
ORF is the segment of DNA between the start codon and the stop codon.
Not all ORFs are translated into proteins, but all CDS are ORFs.
CDS are the "official" recipes that code for proteins.
An operon must have multiple ORFs on the same strand, with the same regulatory set (promoter).

Frameshift Error/Mutation

Occurs due to insertion or deletion of nucleotides that are not multiples of three.
This shifts the reading frame and alters the amino acid sequence encoded by the gene.
Some resistance exists, and the mutated gene may still code for a valid protein, a stop codon may occur early, or the gene may still code for the correct protein due to redundant codons.

ORF ANALYSIS Tools

FramePlot is a graphical analysis tool.
ORFfinder is a tool available at NCBI for identifying all open reading frames.
ORFfinder analyzes provided data and can search databases for sequences.

Example 1: using ORFfinder

This example uses ORFfinder to predict ORFs for the accession number: NM_001185098.2
The following information is required:
- Number of ORFs
- Strand on which ORFs are present
- Frame of the ORFs
- Number of nucleotides and amino acids in the ORFs
- Putative function of the ORFs using BLAST search tool
Remember:
- Strand refers to the plus or minus strand
- Frame refers to +1, +2, +3, -1, -2, -3

ORF Analysis

ORF - Open Reading Frame
ORF - Continuous nucleotide sequence starting with a start codon (ATG) and ending with a stop codon (TAG, TAA, TGA)
ORF - Could be translated into proteins.
ORF - Can be used to predict protein-coding regions but are not a guarantee that the region will be translated.
ORF Finder - Graphical analysis tool available through NCBI that finds all open reading frames in a provided sequence.
ORF Finder Parameters - Minimum length of the predicted ORF (300bp for Homo sapiens), genetic code (standard for eukaryotes), ATG only (excludes shorter non-coding ORFs).
FramePlot - Tool to predict protein-coding regions in DNA, especially those with high G+C content.
FramePlot - Provides a graphical output that distinguishes protein-coding regions from non-coding regions.
FramePlot Rules - Rule 1: the coding ORF has the highest (GC%)
FramePlot Rules - Rule 2 (M-L-H): Medium-low-high GC% pattern:
- On + strand: Dotted line followed by solid line, then dashed line
- On - strand: Dotted line followed by dashed line, then solid line
Frameshift Mutation - Insertion or deletion of nucleotides not in multiples of three.
Frameshift Mutation Outcome - Incorrect codon reading leading to different amino acids.
Frameshift Mutation Possibilities -
- Still coding for a valid protein despite mutation
- Early stop codon
- Correct protein due to multiple codons for the same amino acid

CDS and ORF

CDS - Coding Sequence
CDS - Real part of the gene that translates into a protein.
ORF - Segment of DNA between the start and stop codons.
All CDS are ORFs, but not all ORFs are CDS.

Operon

Operon - A group of genes that share the same regulatory set (promoter) and are transcribed together.
ORFs - To be part of an operon, they must be on the same strand and have the same regulatory set.

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Description

This quiz covers the fundamentals of Open Reading Frames (ORFs) and their role in identifying protein-coding genes within genomes. It explores reading frames, the significance of start and stop codons, and the process of in silico gene prediction. Test your knowledge on how annotation adds functional insights to genomic sequences.