Lecture 29

Question 1

Measuring gene expression:

Answer 1

• Can be measured at various levels (and spatially and temporally)
• Changes in expression can also be detected

Question 2

The phenotypic approach of understanding gene function:

Answer 2

• Forward genetics
• Identified genes by isolating mutants
• Clone the genes and characterise them

Question 3

The candidate approach of understanding gene function:

Answer 3

• 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

Question 4

Identifying interesting mutants (phenotypic approach):

Answer 4

• 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

Question 5

Identifying interesting mutants (candidate approach):

Answer 5

• 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

Question 6

Cloning by homology (good for short genes):

Answer 6

• 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

Question 7

Identifying conserved motifs and using homology:

Answer 7

• 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

Question 8

Annotating genes:

Answer 8

• ## Compare genomic with cDNA to identify introns and exons 5’ UTRS and polyA sites

Question 9

Gene organisation:

Answer 9

• 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

Question 10

Determining gene structure:

Answer 10

• 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

Question 11

cDNA:

Answer 11

• 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

Question 12

RT-PCR:

Answer 12

• 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!

Question 13

RACE:

Answer 13

• 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

Question 14

EST libraries:

Answer 14

• 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

Question 15

Predicting ORFs:

Answer 15

• 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

Question 16

Predicting ORFs for a single gene:

Answer 16

• Is this segment of DNA coding?
• What is it’s reading frame?
• Search for consensus splice signals, stop codons, conserved domains/motifs,
• Either search databases of the sequence, or databases of similar organisms
• When aiming to identify function we can use conserved domains or motifs between organisms that show sequence homology
• Mis-annotations can be identified