DNA sequencing Flashcards
learning objective - outline the steps in automated DNA sequencing via the dideoxy method
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dideoxy sequencing
a method of DNA sequencing that uses dideoxycytidine ribonucleotides to terminate the growth of DNA strands
what is the importance of dideoxyribonucleotides?
chemists can synthesize dideoxyribonucleotides (ddNTPS) that are missing the OH group at the 3’ position
what do ddNTPS tell us?
Sager reasoned that if ddNTP is added to a growing DNA strand, the strand can no longer go due to lack of the OH group
what is the incorporation of ddNTP into a strand called?
chain termination
automated DNA sequencing
each type of ddNTP is labeled with a different fluorescent dye
How much of the starting DNA do we need for automated DNA sequencing?
DNA to be sequenced is obtained in large amounts, which is done by gene cloning
what is the annealing site?
the annealing site is the place in the vector where a primer will bind, which is next to the target DNA that was inserted
what is the purpose of automated DNA sequencing?
determine the base sequence of the target DNA next to the annealing site
what are the steps of Sanger sequencing?
1) a sample with many copies of the single stranded vector is mixed with primers
2) primer bonds to DNA at annealing site
3) dNTPs are added at high concentrations
4) ddNTPS are added at low concentration
5) DNA polymerase is added
sequence ladder
a series of bands on a gel that can be followed in order to determine the base sequence strand of DNA
gene mutagenesis
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learning objective
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learning objective
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why is studying mutations important?
geneticists have discovered that many inherited human diseases, like sickle cell disease, involve mutations within specific genes. This provides insight to the function of genes in unaffected individuals
site directed mutagenesis
allows a researcher t produce a mutation at a specific site within a cloned DNA segment
how is site directed mutagenesis useful?
it can be introduced into a living organism to see how much the mutation affects the expression of a gene, the function of a protein, and the phenotype of the organism
why is site directed mutagenesis sometimes called oligonucleotide directed mutagenesis?
an oligonucleotide primer anneals to the template DNA, where the base sequence of this primer is designed. The primer contains a mismatched region that is not complimentary to the DNA, which is where the mutation occurs
what components are needed for site directed mutagenesis?
dNTPs, DNA polymerase, DNA ligase
what is the final product of this sequencing?
a sequence with a designed mutation that is inserted into bacteria for further investigation
crispr-cas technology
a method that uses the components of the crispr-cas system of genome defense found in prokaryotes to introduce mutations into genes
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21.5 learning objective
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