Isolation of genes Flashcards
What are the three ways genes can be isolated?
- reverse transcriptase
- sticky ends/restriction endonucleases
- gene machine
What is the 1st step in gene isolation using reverse transcriptase?
- mRNA taken from cell that produces protein is used as a template
What is formed in the presence of reverse transcriptase in gene isolation using reverse transcriptase?
cDNA (complementary DNA)
What is the role of DNA helicase in gene isolation using reverse transcriptase?
hydrolyses the mRNA and cDNA, leaving exposed complementary bases on the cDNA.
What is occurs when DNA polymerase is present in gene isolation using reverse transcriptase?
a complementary strand of bases is formed, therefore there is DNA of the desired gene
What are restriction endonucleases?
enzymes that can cut DNA at a specific base sequence
What is the site where restriction endonucleases can cut DNA called?
recognition/restriction site
What is the cleavage site (in gene isolation by restriction endonucleases)?
the site where the enzyme cuts, between specific bases
What are sticky ends?
staggered pieces of DNA that are complementary to each other- formed by restriction endonucleases
Why are sticky ends important in DNA technology?
If DNA is cut with the same restriction endonuclease, DNA sequences from different organisms can be joined (complementary base pairing).
What is the role of DNA ligase in gene isolation by restriction endonucleases?
join the sugar-phosphate backbone
What are three advantages of using a gene machine for gene isolation?
- quick
- more accurate
- can be used for any sequence of nucleotides
- no introns
What is the first step of gene isolation using a gene machine?
use the protein to obtain the amino acid sequence
What is the amino acid sequence used for in the isolation of genes using a gene machine?
to find mRNA
What is mRNA turned into, when isolating genes using a gene machine?
strand of DNA
What are oligonucleotides?
short, overlapping, single-stranded sections of DNA
What are oligonucleotides converted in to in the isolation of genes using a gene machine?
a single stranded copy of the desired gene
What does the PCR do?
- creates double-stranded DNA
- replicates the DNA
What is done after the PCR reaction (in gene isolation using a gene machine)?
inserted into a plasmid (see transformation for more info)
Why cant a gene be obtained from the human genome?
- contains introns
- bacteria cannot remove introns
Why is using the gene machine faster?
less steps involved/ quicker than enzyme catalysed reactions
