DNA has a charge associated with it. What functional group that carries a charge is included in DNA, and how do we use the charge to our advantage while doing gel electrophoresis? How does this relate to using the phrase
Asked By Sakshi
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A) DNA has a charge associated with it. What functional group that carries a charge is included in DNA :
Chemical formula of phosphate molecules is PO4-.
Phosphate groups are present in DNA backbone will have negatively-charged oxygen molecules that give DNA an overall negative charge.
B) We use the charge to our advantage while doing gel electrophoresis As it helps in separation of DNA. The DNA is loaded into pre-cast wells in the gel and then apply current. As explained earlier, phosphate backbone of the DNA (or RNA) molecule is negatively charged, so when it is placed in an electric field, then DNA fragments will migrate to the positively charged anode and helps in separation of DNA fragments of varying sizes.
C) This relate to using the phrase “run to the red” while performing gel electrophoresis
After amplification of a specific region of DNA by PCR, gel electrophoresis will allow us to visualize the PCR product(s) and separate DNA fragments based on size. An agarose gel is used. The agarose is similar to gelatinand will form a matrix as it solidifies. After the gel has solidified, it is placed into a box that contains a bufferingsolution. PCR products are loaded into the wells (holes) at one end of the gel, using a pipette. The box is then covered with its lid. The lid contains electrodes, which are then hooked up to a power supply. The phosphate groups on DNA’s sugar-phosphate backbone carry negative charges. Therefore, when the power supply is turned on, the electrical current will pass through the buffer and the DNA will be attracted to the positiveside (red electrode). The smallerthe DNA fragment, the fasterit will move (“run”) through the gel. The DNA fragments are then visualized by using Ethidium Bromide in the gel to “stain” DNA. This stain emits orange lightwhen illuminated by UV. Use extreme caution when handling the gel, as ethidium bromide is a potential carcinogen.
D) What holds the two strands of a DNA molecule together?
The two strands of a DNA molecule are held together by hydrogen bonds between bases on opposite strands.
E) This attachment affected by the addition of heat...
As adenine pair with thymine A-T by two hydrogen bonds, therefore, takes less heat energy to denature. Guaninine pairs with cytosine G-C by three hydrogen bonds so takes more energy to break the bonds between them.
During heating, heat can pull the two strands of DNA apart. This happens at critical temperature Tm, process is called 'denaturation'; to separate the strands.
The two strands are complementary so, if we cool the then they stick tightly, reforming double-stranded DNA, process is called 'annealing' or 'hybridization', as specific; so only complementary strands bind.
So to conclude, when DNA solution is heated enough, the double-stranded DNA unwinds and the hydrogen bonds holding two strands together weaken and break. The amount of strand separation measured by the absorbance of the DNA solution at 260nm....
- 09 Aug 2018 @ 02:28