PTC genotyping Flashcards
what was the purpose of this experiment?
cheek cell DNA will be amplified by PCR at the TAS2R38 locus. The PCR product will be used in genotyping
what is polymerase chain reaction used for?
it’s an in vitro method of DNA replication used to amplify selected DNA sequences
what enzyme is used in the PCR reaction?
DNA polymerase
what is used instead of primase?
a pair of specific primers will bind to a different end of the region to be amplified
how much DNA can be replicated?
usually less that 1kb
why can only less than 1kb be replicated?
there’s no ligase to link Okazaki fragments
what is used instead of helicase?
heat is used to separate the strands of DNA (denaturing)
how do we prevent the DNA polymerase from being denatured by the heat? (ie, which type do we use?)
PCR uses a special heat resistant form of DNA polymerase isolated from thermophilic bacteria called Taq polymerase
what substrates are used in PCR?
deoxyribonucleotide triphosphates (dNTPs)
what is used as a cofactor
Mg2+ ions as a cofactor for the polymerase
when DNA is denatured by heat, what is the result?
single stranded DNA molecules
what happens as the temp is lowered?
the two primers hybridize to their complementary sequences in the single stranded DNA
what does the DNA pol do?
it replicates the single-stranded DNA segments from extending from each primer, forming 2 double stranded DNA molecules identical to the parental molecule
where were the cells from that we used in this lab?
cheek cells
how where the cheek cells obtained?
saline mouthwash
the DNA from these cells was heated in a tube containing ___
Chelex resin
what is the purpose of the Chelex resin?
the near boiling temp. will lyse the cell membrane, DNA and other cell contents will be released, and the Chelex will protect the DNA
what was the isolated DNA added to?
Ready-to-go PCR beads
what do the Ready-to-go PCR beads contain?
Taq polymerase, dNTPs, and MgCl2
next, the primer/loading-dye mix was added. What does this contain?
contains the pair of primers, sucrose, and cresol red dye
what is the purpose of the dye?
the dye components allows the amplified product to be directly loaded into an agarose gel for electrophoresis
what does the TAS2R38 gene encode?
the PTC taste receptor (one of the receptor proteins on the surface of a taste cell that recognizes bitterness)
what does RFLP stand for
restriction fragment length polymorphism
what is an RFLP?
differences in DNA sequences between individuals can result in different lengths of cut fragments = RFLPs
what enzyme recognizes a sequence in the TAS2R38 gene?
HaeIII
what sequence does HaeIII recognize?
-GGCC-
what allele contains -GGCC-
the taster allele
what is the non taster allele
-GGGC-
will the non taster allele be cut by HaeIII?
no
how big is a taster allele
221bp
what size fragments will the taster allele be cut into?
177bp and 44bp
how many bands will a taster allele show?
2
how many bands will a non taster allele show?
1
how many bands will a heterozygous person show?
3!
