PTC genotyping

Question 1

what was the purpose of this experiment?

Answer 1

cheek cell DNA will be amplified by PCR at the TAS2R38 locus. The PCR product will be used in genotyping

Question 2

what is polymerase chain reaction used for?

Answer 2

it’s an in vitro method of DNA replication used to amplify selected DNA sequences

Question 3

what enzyme is used in the PCR reaction?

Answer 3

DNA polymerase

Question 4

what is used instead of primase?

Answer 4

a pair of specific primers will bind to a different end of the region to be amplified

Question 5

how much DNA can be replicated?

Answer 5

usually less that 1kb

Question 6

why can only less than 1kb be replicated?

Answer 6

there’s no ligase to link Okazaki fragments

Question 7

what is used instead of helicase?

Answer 7

heat is used to separate the strands of DNA (denaturing)

Question 8

how do we prevent the DNA polymerase from being denatured by the heat? (ie, which type do we use?)

Answer 8

PCR uses a special heat resistant form of DNA polymerase isolated from thermophilic bacteria called Taq polymerase

Question 9

what substrates are used in PCR?

Answer 9

deoxyribonucleotide triphosphates (dNTPs)

Question 10

what is used as a cofactor

Answer 10

Mg2+ ions as a cofactor for the polymerase

Question 11

when DNA is denatured by heat, what is the result?

Answer 11

single stranded DNA molecules

Question 12

what happens as the temp is lowered?

Answer 12

the two primers hybridize to their complementary sequences in the single stranded DNA

Question 13

what does the DNA pol do?

Answer 13

it replicates the single-stranded DNA segments from extending from each primer, forming 2 double stranded DNA molecules identical to the parental molecule

Question 14

where were the cells from that we used in this lab?

Answer 14

cheek cells

Question 15

how where the cheek cells obtained?

Answer 15

saline mouthwash

Question 16

the DNA from these cells was heated in a tube containing ___

Answer 16

Chelex resin

Question 17

what is the purpose of the Chelex resin?

Answer 17

the near boiling temp. will lyse the cell membrane, DNA and other cell contents will be released, and the Chelex will protect the DNA

Question 18

what was the isolated DNA added to?

Answer 18

Ready-to-go PCR beads

Question 19

what do the Ready-to-go PCR beads contain?

Answer 19

Taq polymerase, dNTPs, and MgCl2

Question 20

next, the primer/loading-dye mix was added. What does this contain?

Answer 20

contains the pair of primers, sucrose, and cresol red dye

Question 21

what is the purpose of the dye?

Answer 21

the dye components allows the amplified product to be directly loaded into an agarose gel for electrophoresis

Question 22

what does the TAS2R38 gene encode?

Answer 22

the PTC taste receptor (one of the receptor proteins on the surface of a taste cell that recognizes bitterness)

Question 23

what does RFLP stand for

Answer 23

restriction fragment length polymorphism

Question 24

what is an RFLP?

Answer 24

differences in DNA sequences between individuals can result in different lengths of cut fragments = RFLPs

Question 25

what enzyme recognizes a sequence in the TAS2R38 gene?

Answer 25

HaeIII

Question 26

what sequence does HaeIII recognize?

Answer 26

-GGCC-

Question 27

what allele contains -GGCC-

Answer 27

the taster allele

Question 28

what is the non taster allele

Answer 28

-GGGC-

Question 29

will the non taster allele be cut by HaeIII?

Answer 29

no

Question 30

how big is a taster allele

Answer 30

221bp

Question 31

what size fragments will the taster allele be cut into?

Answer 31

177bp and 44bp

Question 32

how many bands will a taster allele show?

Answer 32

2

Question 33

how many bands will a non taster allele show?

Answer 33

1

Question 34

how many bands will a heterozygous person show?

Answer 34

3!