Lecture 29 Flashcards
1
Q
Measuring gene expression:
A
- Can be measured at various levels (and spatially and temporally)
- Changes in expression can also be detected
2
Q
The phenotypic approach of understanding gene function:
A
- Forward genetics
- Identified genes by isolating mutants
- Clone the genes and characterise them
3
Q
The candidate approach of understanding gene function:
A
- Reverse genetics
- Identify genes based on their predicted function
- Clone the genes and characterise them
- Limiting as it requires prediction of what it is involed
4
Q
Identifying interesting mutants (phenotypic approach):
A
- Clone genes using
- Positional cloning: determine position of the gene, generate a physical map and use chromosome walking
- Complementation: DNA mediated transformation if you have the right organism
- Insertional mutagenesis: Tag genes by insertion of DNA then screen for phenotypes
5
Q
Identifying interesting mutants (candidate approach):
A
- Clone the genes
- Candidate genes selected on the basis of the encoded gene product
- Information already obtained can help, and massive technical sequencing capacity now exists
6
Q
Cloning by homology (good for short genes):
A
- If your organism has been sequenced, clone the gene by PCR
- Identify probable homologue by search databases for similarity and use databases as subjects
- If your organism doesn’t have a sequence, sequence its genome then clone by PCR, identify genes of interest in related organisms, using conservation
7
Q
Identifying conserved motifs and using homology:
A
- Clone the gene of interest, and identify genes in other organisms that have homology with our gene of interest
- Back-translate protein to DNA
- Design degenerate PCR primers
- Restriction nuclease digestion and DNA cloning, probe the library using your gene of interest and identify where your gene of interest is found
8
Q
Annotating genes:
A
- ## Compare genomic with cDNA to identify introns and exons 5’ UTRS and polyA sites
9
Q
Gene organisation:
A
- One gene can encode multiple transcripts (mRNA) which can encode multiple proteins (multiple start sites, polyA sites and splicing)
- The number of genes doesn’t necessarily correlate with complexity
- Different gene expression is also a factor to consider
10
Q
Determining gene structure:
A
- Transcript mapping via PCR and RT-PCR, Rapid amplification of cDNA ends (RACE), comparison of genomic and cDNA
- Identify open reading frames from consensus sequences or conservation
11
Q
cDNA:
A
- ds cDNA is a copy of the original mRNA
- hybridise mRNA with polyT primers
- Make complementary DNA copy with RTs
- Degrade RNA with RNase
- Synthesise a second cDNA strand using DNA polymerase
- The RNA fragment acts as a primer
- can work out the intron sites, 3’ end and polA tail
12
Q
RT-PCR:
A
- Based on comparing the product of PCR reactions
- PCR mRNA and the genomic sequences
- the Gene produce includes intron sequences
- The mRNA produce lacks intron sequences
- The size different indicates the size of the intron, and lining them up can determine the location of the intron!
13
Q
RACE:
A
- Working out the 5’ and 3’ ends
- Reverse transcription reaction using a gene specific primer
- Use another nested primer to amplify the fragment of interest
- Use PCR
- This can be done one at a time, or all at once
- Random primers can be used to create short fragments of every transcript
14
Q
EST libraries:
A
- Matures transcripts that cover the entire genome
- Random primers are used so short fragments of every part of the genome will be able to be generated into a library
15
Q
Predicting ORFs:
A
- Know the direction, so only need 3 frames
- Look for ATG’s that could be the start
- Look for termination codons
- Find blocks that create proteins, and see what kind of protein this will create
- Not ideal for an entire genome, but ok for a single gene
