Loading....
Coupon Accepted Successfully!

Biochemistry

Open Flashcards

Purines, Pyrimidines and Nucleic Acid Metabolism

Question
19 out of 59
 

Following are required for PCR except (AIIMS Nov 2010)



A Deoxyribonucleotides
B Taq polymerase

C Dideoxyribonucleotides
D Template DNA

Ans. C Dideoxyribonucleotides

Polymerase Chain Reaction

a. The polymerase chain reaction (PCR) is a test tube method for amplifying a selected DNA sequence that does not rely on the biologic cloning method described on p. 467. PCR permits the synthesis of millions of copies of a specific nucleotide sequence in a few hours.

b. It can amplify the sequence, even when the targeted sequence makes up less than one part in a million of the total initial sample. The method can be used to amplify DNA sequences from any source—bacterial, viral, plant, or animal.

Steps of a PCR

PCR uses DNA polymerase to repetitively amplify targeted portions of DNA. Each cycle of amplification doubles the amount of DNA in the sample, leading to an exponential increase in DNA with repeated cycles of amplification. The amplified DNA sequence can then be analyzed by gel electrophoresis, Southern hybridization, or direct sequence determination.

a. Primer construction: It is not necessary to know the nucleotide sequence of the target DNA in the PCR method. However, it is necessary to know the nucleotide sequence of short segments on each side of the target DNA.

b. These stretches, called flanking sequences, bracket the DNA sequence of interest. The nucleotide sequences of the flanking regions are used to construct two, single-stranded oligonucleotides, usually 20–35 nucleotides long, which are complementary to the respective flanking sequences.

c. The 3′-hydroxyl end of each primer points toward the target sequence (see Figure 33.19). These synthetic oligonucleotides function as primers in PCR reactions.

d. Denature the DNA: The DNA to be amplified is heated to separate the double-stranded target DNA into single strands.

e. Annealing of primers to ssDNA: The separated strands are cooled and allowed to anneal to the two primers (one for each strand).

f. Chain extension: DNA polymerase and deoxyribonucleoside triphosphates (in excess) are added to the mixture to initiate the synthesis of two new chains complementary to the original DNA chains.

g. DNA polymerase adds nucleotides to the 3′-hydroxyl end of the primer, and strand growth extends across the target DNA, making complementary copies of the target.

h. [Note: PCR products can be several thousand base pairs long.] At the completion of one cycle of replication, the reaction mixture is heated again to denature the DNA strands (of which there are now four).

i. Each DNA strand binds a complementary primer, and the cycle of chain extension is repeated. By using a heat-stable DNA polymerase (for example, Taq polymerase) from a bacterium (for example, Thermus aquaticus) that normally lives at high temperatures (a thermophilic bacterium), the polymerase is not denatured and, therefore, does not have to be added at each successive cycle.

j. Typically 20–30 cycles are run during this process, amplifying the DNA by a million-fold to a billion-fold. [Note: Each extension product of the primer includes a sequence complementary to the primer at the 5′-end of the target sequence (see Figure 33.19). Thus, each newly synthesized polynucleotide can act as a template for the successive cycles (see Figure 33.20). This leads to an exponential increase in the amount of target DNA with each cycle hence the name “polymerase chain reaction.”]

k. Dideoxyribonucleotideslook exactly like deoxyribonucleotides except that they no OH group on the third carbon of the sugar.

l. This has very serious implications. These nucleotides can add to the growing DNA chain (because they have a phosphate on their fifth carbon that can link to the hydroxyl on the third carbon of the nucleotide at the end of the growing chain) but once on, no additional nucleotides can be added.

m. In other words, dideoxyribonucleotides act as chain terminators. When they are incorporated, the polymerization ends. It is the ability to end chains with these molecules that is the basis for DNA sequencing by the Sanger procedure.

Purines, Pyrimidines and Nucleic Acid Metabolism Flashcard List

59 flashcards
1)
2)
3)
4)
5)
6)
7)
8)
9)
10)
11)
12)
13)
14)
15)
16)
17)
18)
19)
20)
21)
22)
23)
24)
25)
26)
27)
28)
29)
30)
31)
32)
33)
34)
35)
36)
37)
38)
39)
40)
41)
42)
43)
44)
45)
46)
47)
48)
49)
50)
51)
52)
53)
54)
55)
56)
57)
58)
59)