Chapter 21.1 Flashcards
learning objective 1 - outline the procedure for cloning a gene into a vector
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learning objective 2 - describe how cDNA is made
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learning objective 3 - compare and contrast a genomic library with a cDNA library
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how is researching proteins compared to finding a needle in a hay stack?
because in all species, any given cell can express hundreds or thousands of different proteins
what is gene cloning?
refers to the process of making copies of a gene, which can be done through techniques like PCR or introduction of a gene into a vector
what are the benefits of DNA sequencing?
cloned genes provide enough DNA for sequencing, which helps find the sequence of a gene’s promoter, regulatory sequence, and coding sequence, as well as identifying alleles that can cause cancer and disease
what is a vector?
a small segment of DNA that a gene is inserted into
When is a vector used?
When a gene is removed from its native site within a chromosome, it is inserted into vector
what is the function of a vector?
it can function and replicate independently of chromosomal DNA and produce copies, so it can act as a carrier of the DNA segment that is going to be cloned.
where is a vector replicated?
a vector is replicated within a living cell, also called the host cell. when the vector is replicated, so is the DNA it holds
where are vectors commonly derived from?
plasmids or viruses
most vectors are ____
plasmids
what is a plasmid?
small circular pieces of DNA
what kind of genes to plasmids carry?
plasmids tend to carry genes that confer resistance to antibiotics or other toxic substances - these are called R factors
what else do plasmids carry?
they also contain an origin of replication sequence
what does the origin of replication sequence within a plasmid determine?
whether or not the vector can replicate within a particular type of host cell
while some plasmids have a _______ range of origins of replication, many vectors have _____ range
broad, limited
some plasmids can make ______ copies, while others can only make ____ or _____ copies
100-200, one or two
what is a selectable marker?
when the expression of the gene selects for the growth of the host cells, such as the ability to grow in toxic environments
how do viral vectors work?
because viruses infect living cells and propagate within them, a gene can be inserted into a viral genome, where the gene is then replicated once the viral DNA is replicated
what is used to clone large pieces of DNA?
cosmids, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs)
how does DNA get inserted into a vector?
by cutting and pasting of the DNA fragments using enzymes called restrictions endonuclease/restriction enzymes
how do restriction enzymes work?
the restriction enzymes bind to a specific base sequence, then cleave the DNA backbone at two locations
what are sticky ends?
single stranded DNA regions, which holds DNA strands together
what are sticky ends for?
two dna sequences that have been cut will have sticky ends, which will be put together to form one larger DNA sequence. dna ligase forms hydrogen bonds and puts them back together
palindromic
the sequence in one strand is the same as that in the complimentary strand when read in the opposite direction
