L15 Classical Gene Cloning 2 Flashcards
(61 cards)
What is a genomic library?
A collection of recombinant clones that together cover the entire genome of an organism.
What organism is typically used as the host for a genomic library?
E. coli
What is inserted into cloning vectors to create a genomic library?
fragments of genomic DNA
What is a recombinant clone?
a bacterial cell carrying a unique recombinant vector with a distinct genomic DNA insert
How can a genomic library be stored for future use?
by freezing the bacterial cells
How are specific clones selected from a genomic library?
based on the DNA sequence inserted
What tool is commonly used to cut genomic DNA for cloning?
Restriction enzymes
Why are 6-cutter enzymes often used for genomic libraries?
they produce fragments ~4,000 bp in length, ideal for capturing whole genes
Why is a 4,000 bp fragment size ideal for cloning bacterial genes?
It’s long enough to include whole genes and promoter sequences
What is a downside of DNA fragments being too large?
it reduces ligation efficiency
What’s the benefit of sticky ends over blunt ends in ligation?
sticky ends facilitate hybridization and increase ligation efficiency.
Why might someone still choose blunt ends for cloning?
no need for matching restriction enzymes
What does it mean if two restriction enzymes have compatible sticky ends?
their DNA cut fragments can be ligated together despite different recognition sites
Name two enzymes with compatible sticky ends due to overlapping recognition sequences.
BamHI and Sau3A
What is the recognition sequence length for Sau3A?
every 256 bp
Why must insert DNA size be optimized when making a genomic library?
Too small = gene fragmentation, too large = inefficient ligation
What determines the location of cut sites in a restriction digest?
the random distribution of recognition sequences
Why do we sometimes need multiple genomic libraries?
To ensure any gene of interest is captured in at least one perfect sized fragment
What is partial digestion?
using a restriction enzyme briefly so it only cuts some of the recognition sites
Why does a partial digest produce a smear on a gel?
Because different genome copies are cut at different positions
Which enzyme is typically used for partial digestion in library creation?
Sau3A
How can you ensure gene X is always present in a genomic library?
by partially digesting the DNA so fragments overlap
What physical method mimics the effect of partial digestion?
DNA shearing through a fine needle. The finer the needle the more smeared it would look (i.e. the size gets smaller)
What is a key difference between partial digestion and shearing?
Shearing produces blunt ends, partial digestion with restriction enzymes like Sau3A creates sticky ends
