Gene Identification and Analysis Flashcards
What 2 ways can we find genes in the nucleotide sequence?
How do these work?
1) Prediction software
- Scans the sequence for promoters, start + stop sequences and intron splice sites
2) Computer analysis to translate the DNA in the 6 reading frames (3 reading frames in each direction of DNA)
- THEN, search for similarities to known proteins, using BLAST software
What is the disadvantage of using prediction software to find genes in the nucleotide sequence?
Can not be sure if they are true
What is BLAST software?
What is the process using this?
Searches for similarities to KNOWN proteins:
1) Input amino acid sequence of the proposed protein (6 reading frames)
2) BLAST tries to align the unknown protein sequence to ALL known proteins in the database - searching for proteins with similar sequences
3) Produces a report of alignment of the proposed protein to known protein.
- Shows how many amino acids are the same and in the same order
- Show similar charges
What does it mean if there is similarity found between the proposed protein and a known protein?
- Suggests that the proteins have evolved from the same common ancestor
- Suggests they have similar molecular functions (eg. if known is a transcription factor, proposed may be too)
What is Vega and what is it used for?
Used to look at the genome online - to identify if there is any characteristics of a gene in that DNA sequence (prediction software)
(looks for promoters, intron splice sites etc.)
What are Ests and how are they expressed?
Expressed Sequence Tags
Short sequences at the end of cDNA
Expressed as RNA
When using the prediction software, can one piece of evidence (eg. intron splice site) be used as stand-alone evidence to say that there is a gene there?
NO, multiple parts of evidence must be used together
What are microarrays used for?
To identify genes
Allow to compare the TRANSCRIPTIOMES of different tissues
- In different states (eg. normal and cancerous)
- In different tissues (liver, spleen)
What are the advantages of microarrays?
High throughput - small scale, fast, automated
Describe the process of a microarray, to discover which genes are turned on in a specific tissue
1) Grid formed - each position in the grid contains ONE cDNA
2) Purify mRNA from a tissue and tag it with a fluorescent dye
3) Add the mRNA in solution, onto the array - allowing the mRNA to hybridise and rinse off the excess (removes any unbound DNA)
4) If the mRNA hybridises to any cDNA in the grid - shows that the genes are present
5) Use a sensitive camera to detect which genes are ‘ON’
6) Can compare to the microarray of a diseased tissue to discover which genes are up-regulated/ down-regulated
What carries out the microarray process?
A very precise robot - manufactures the array on a microscopic grid
What is cDNA?
What are its features?
Complimentary DNA (complementary to mRNA) Single stranded
Synthesised from a single strand of RNA
Contains only EXONS
Antisense DNA strand
Made by reverse transcriptase
When comparing microarrays between a normal tissue and one of a tumour, what POTENTIALLY are the genes that are LOST in the tumour?
POTENTIALLY tumour suppressor genes
When comparing microarrays between a normal tissue and one of a tumour, what POTENTIALLY are the genes that are GAINED in the tumour?
POTENTIALLY oncogenes
When comparing microarrays between a normal tissue and one of a tumour, what are the genes that are THE SAME in the tumour?
House-keeping genes and are specific to that tissue
From a microarray, how can the identity of the genes be found?
Using the grid coordinates
What 3 ways can genes be identified?
1) By making a cDNA library from mRNA (represents the transcriptome
2) By making a genomic library and then make PREDICTIONS based upon the genomic sequence using BLAST
3) Using microarrays
What are the different ways of analysing the function of the identified gene?
1) Reverse genetics in mice
2) Forward genetics
What must be done before forward/reverse genetics can be used to analyse the function of a gene?
- Must have already made PREDICTIONS of the function (eg. using microarrays and BLAST)
- Forward/reverse genetics BACKS up the prediction
What are the disadvantages of genetic engineering in mice?
Time consuming and expensive
What must you start with in order to genetically engineer mice?
A GENOMIC clone of the endogenous gene
What are the 2 approaches to genetic engineering in mice?
Describe them
1) Gene Replacement (Altering the gene)
- Make small changes to the endogenous gene
2) Gene Knock-out (Destroying the gene)
What does gene knock-out determine?
The function of the gene (if remove the gene - no longer have the function)
What is the process of gene knock-out in mice? (of a single gene)
1) Insert 2 mammalian genes into the genomic clone of the gene
- NEO inserted directly into the exon of the genomic clone
- TK placed off to one side of the exon
2) Purify the DNA from the bacteria
3) Introduce the construct into mouse EMBRYONIC stem cells (ES) using cell culture techniques
4) Some of the DNA makes it into the nucleus of the mouse and into the MOUSE GENOME
In gene knock-out, what are ‘homologous arms’?
Gene sequences that flank NEO on either side in the GENOMIC CLONE
Have homology to the mouse genome
In gene knock-out, what is recombination of the construct into the genome of the mouse driven by and how?
Is this efficient?
The ENDOGENOUS DNA repair machinery
Identifies fragments of DNA swimming around the cell and tries to put things back together
NOT efficient
In gene-knock out, what 2 things can occur when the construct is integrated into the genome?
Which occurs more frequently?
1) HOMOLOGOUS recombination
2) NON-HOMOLOGOUS recombination
Occurs more frequently
Describe homologous recombination of the construct into the mouse genome
- DNA repair mechanism recognises the HOMOLOGOUS ARMS flanking NEO
- Inserts the construct into the homologous gene - forms a transgene
- Endogenous gene is knocked out
- TK is lost