MolBio12-13 - 42 Flashcards
Characterise restriction enzymes
Dimers that recognize precise palindromic DNA sequences, either cutting with overhangs, or flush (blunt)
How do restriction enzymes work?
In a small volume of buffered water at 37C for 2 hours
Where do restriction enzymes come from?
Identified and purified from bacteria
Why did restriction enzymes evolve?
To prevent entry of foreign DNA into the cell
How are restricted DNA fragments seperated?
PAGE
Name a dye used in SDS-PAGE to stain DNA
Ethidium bromide
What are cohesive termini?
Restricted overhangs that will bind
Name two compatible restriction overhangs
XbaI and SpeI
What is the XbaI overhang?
aGATC
What is the SpeI overhang?
tGATC
What are plasmid vectors?
Small, circular extra-chromosomal DNA that occur naturally in bacteria
What is stereotypical of plasmid vectors?
Their own origin of replication - results in around 50 copies produced; usually carry antibiotic resistance genes
How much information do plasmid vectors usually hold?
<30kB of DNA
What is transformation?
Mixing of bacteria with plasmids, then creating temporary holes for their uptake through the membrane
What is the problem with transformation?
Not very efficient
How is the relative inefficiency of transformation overcome?
Antibiotic selection
What is a cDNA library?
Library of DNAthat codes the transcriptome
What is a genomic library?
Library of all DNA
What is the advantage to a cDNA library?
Easy to compare what genes are expressed in pathological vs healthy tissue
What is the advantage to a genomic DNA library?
Contains all regulatory sequences, allowing study of transcriptional regulation
What are ESTs?
Expressed sequence tags
What do ESTs do?
Ends all clones in the cDNA library - useful in genetic engineering
Outline dideoxy terminator sequencing
Denature template, cool with primer, start DNA synthesis with DNA polymerase and tagged ddNTPs. DNA polymerase cannot extend beyond ddNTP, allowing identification of which ddNTP is at that position
How can ddNTP sequencing be read?
Film (100-400 NT); automation by camera (<1000 NT)
What is progressive sequencing?
Sequence from either end, restarting each time you hit about 1000 NT, until you meet in the middle
What is shotgun sequencing?
Sequence loads of short random sequences (traces) and let a computer assemble them into a contig
What is the advantage of shotgun sequencing?
No need for thought
What is the disadvantage of shotgun sequencing?
Need to sequence more than 6x the size of the genome
What is parallel sequencing?
Combination of shotgun and progressive
When was the human genome sequenced?
1990-2003
How long does it currently take to sequence a genome?
56 hours
How are genes found?
Gene prediction software, or using a computer to translate all 6 reading frames
How does gene prediction software function?
Looks for start/stop/splice sites
Outline Blast protein alignment
Input a/a sequence of proposed protein, Blast searches for other proteins with similar sequences, which shows alignment of query with subject
What does a similarity Blast result tell us?
Possibility that proteins evolved from same common ancestor and have similar molecular proteins
What is a microarray?
Sample of 10,000s genes at once - small scale, fast and automated, each spot containg one cDNA
In what three ways are genes ID’d?
cDNA library, genomic predictions, microarray interest
How can genes be added to mice?
Homologous and non-homologous recombination
What is homologous recombination?
Only gene with homologous arms added - nothing else
What is non-homologous recombination?
Cell’s DNA machinery recombines the construct (very inefficient)
How are genes knocked out in mice?
Add NEO/TK complex in gene (making it not work); treat with neomycin (positive selector for NEO); treat with GANC (kills all with TK); cells introduced into mice: 1st gen = mosaic; 2nd gen = non-mosaic carriers; 3rd gen = mixture of homozygous KO and wild
