Why NaOH Hydrolyzes RNA in Northern Blot: The Role of the 2′-Hydroxyl Group
The procedure of Southern blotting involves treatment of the solid support (nitrocellulose) containing the DNA with NaOH to denature the double helix. Treatment of a Northern blot with NaOH, however, will lead to the hydrolysis of the nucleic acid on the filter paper. This is due to which major chemical feature of the nucleic acids involved in a Northern
blot?
A, The presence of thymine
B. The presence of uracil
C. The presence of a 2-hydroxyl group
D. The presence of a 3′-hydroxyl group
Understanding RNA Hydrolysis in Northern Blotting
In Southern blotting, DNA is transferred onto a solid support, typically nitrocellulose, and treated with NaOH to denature the double-stranded helix. This allows single-stranded DNA probes to hybridize effectively. However, when NaOH is applied in a Northern blot, it leads to the hydrolysis (degradation) of RNA instead of simple denaturation.
The reason for this lies in the chemical structure of RNA, specifically the presence of a 2′-hydroxyl (-OH) group (Option C), which is absent in DNA.
Why Does NaOH Hydrolyze RNA But Not DNA?
-
RNA Structure vs. DNA Structure:
- RNA contains ribose sugar, while DNA contains deoxyribose (which lacks an -OH at the 2′ position).
- The 2′-hydroxyl group (-OH) in RNA makes it chemically unstable under alkaline (NaOH) conditions.
-
Mechanism of Alkaline Hydrolysis:
- Under basic conditions, the 2′-OH group attacks the adjacent phosphate group, leading to cleavage of the RNA backbone into small fragments.
- DNA, lacking the 2′-OH, remains intact under the same conditions.
Why Are the Other Options Incorrect?
-
A. The Presence of Thymine
- Thymine is found in DNA, not RNA, and does not contribute to alkaline hydrolysis.
-
B. The Presence of Uracil
- While uracil is unique to RNA (instead of thymine), it does not affect RNA’s susceptibility to NaOH degradation.
-
D. The Presence of a 3′-Hydroxyl Group
- Both RNA and DNA have a 3′-hydroxyl group, but only RNA is hydrolyzed by NaOH due to the additional 2′-OH group.
Conclusion:
The 2′-hydroxyl group in RNA makes it highly sensitive to alkaline hydrolysis, preventing the use of NaOH for denaturation in Northern blotting. Instead, formamide or heat is typically used for RNA denaturation.
Understanding these structural differences between DNA and RNA is essential for molecular biology research and blotting techniques!
69 Comments
Suman bhakar
March 3, 2025👍👍
Ujjwal
March 3, 2025👍
Arushi
March 3, 2025👍👍
Akshay mahawar
March 3, 2025Done 👍
Priyam choudhary
March 3, 2025Done
Priti khandal
March 3, 2025Okk sir
Nisha
March 4, 2025Ok
Prami Masih
March 4, 2025Very nice sir
Abhilasha
March 7, 2025Done sir
Dipti Sharma
August 20, 2025Only RNA is hydrolyzed by NaOH due to the additional 2′-OH group so correct answer is c
Shivani
August 20, 2025Option C is correct
The aditional 2′ OH group makes RNA unstable in alkaline condition
Khushi Mehra
August 20, 2025RNA is hydrolyzed by NaOH due to presence of 2′ OH group in RNA which make it chemically unstable in alkaline condition.
In DNA, 2′ OH is absent, only H is present.
Mohd juber Ali
August 20, 2025Option c
HIMANI FAUJDAR
August 20, 2025Ans (c) Presence of 2 hydroxyl gp in RNA makes it highly sensitive in alkaline condition.
Dharmpal Swami
August 20, 20252’oH absent in dna
Sakshi yadav
August 20, 2025Presence of 2’OH gp in RNA
Varsha Tatla
August 20, 20252′-oH -nt in dna
Mansukh Kapoor
August 20, 2025The correct answer is option 3rd
Because in DNA 2′ OH group is absent and in RNA it is present so it affects the alkality of it.
Surbhi Rajawat
August 20, 2025The NaOH can’t be used in northern blot because it will hydrolyse the 2’OH group present in Ribose sugar. Which is not the case with DNA
Aman Choudhary
August 20, 2025Option c is correct
Because DNA lack oH group on 2 nd position but in RNA 2′-hydroxyl group is present which make RNA unstable in alkaline conditions
Priya choudhary
August 20, 2025Option c is correct
The presence of a 2′-hydroxyl group
Priya choudhary
August 20, 2025Option c is correct,the presence of a 2′-hydroxyl group
Deepika Sheoran
August 20, 2025Option c is correct answer
Because DNA 2OH group is absent &RNA Highly sensitive in Alkaline condition.
Priya dhakad
August 20, 2025Correct option is c
2-hydroxyl group is present in RNA makes its highly sensitive in alkaline condition but absent in DNA
Neelam Sharma
August 20, 20252 hydroxyl grp in RNA do highly sensitive in alkaline condition
Mitali Saini
August 21, 2025C. The presence of a 2-hydroxyl group
Santosh Saini
August 21, 2025Option C is correct ( presence of a 2 hydroxyl group)
Devika
August 21, 2025Option c
Khushi Pareek
August 21, 20252’OH group
Khushi Vaishnav
August 21, 20252′-hydroxyl group (-OH)
Sakshi Kanwar
August 21, 2025Option C as DNA is double stranded and providing NaOH makes it single stranded but in case of RNA the 2′-OH group makes RNA unstable and cleaves the bond and degrade the RNA
Simran Saini
August 21, 2025The presence of a 2′- hydroxyl group.
Anurag Giri
August 22, 2025The answer is option 3rd
Because in DNA 2′ OH group is absent and in RNA it is present so it affects the alkality of it.
Sneha Kumawat
August 22, 2025RNA is hydrolyzed by NAOH due to presence of 2 -oH group
Soniya Shekhawat
August 22, 20252 ‘OH of RNA make it chemically unstable under the alkaline any NAOH condition.
Kirti agarwal
August 23, 2025Presence of 2 hydroxyl group
Priya khandal
August 23, 2025The presence of 3 hydroxyl group
karishma don
August 23, 2025when NAOH is applied to filter paper of northern blotting, there is hydrolysis of RNA due to the presence of OH group on 2′ position of ribose sugar, as in alkaline condition(NAOH) The OH group attacks the adjacent phosphate group and this how cleavage occurs or denaturation occurs of RNA on filter paper.
Akshita Jangid
August 23, 2025Option C is correct
The presence of a 2-hydroxyl group
Bharti Yadav
August 23, 2025RNA is hydrolyzed by NAOH due to presence of 2 -oH group as RNA is highly sensitive to alkaline medium
Kanishka Parnami
August 23, 2025Done
Yogita
August 24, 2025Presence 2-oh groups
Aakanksha Sharma
August 24, 2025Presence of 2-OH group
Himanshi
August 24, 2025the additional 2′-OH group
AKANKSHA RAJPUT
August 24, 20252′-hydroxyl group (-OH)
Bhavana kankhedia
August 24, 2025presence of a 2′-hydroxyl group
Ayush
August 24, 20252′-hydroxyl group This makes RNA unstable in alkaline conditions
Kajal
August 24, 2025Rna is hydrolysed due to presence of additional OH gp at 2nd position…
Tanvi Panwar
August 24, 2025The presence of 2′-hydroxyl group.
Priyanka Verma
August 24, 2025the presence of 2′- hydroxyl group
Parul Yadav
August 24, 2025When NaOH is applied to Northern Blotting it leads to degradation of RNA instead of simple denaturation because of the presence oh 2′-OH group.
Sonam Saini
August 24, 2025Presence of hydrolysis group
sakshi khatri
August 24, 2025c is correct
Surendra Doodi
August 24, 2025One extra OH group make RNA less stable and more reactive
Kajal
August 25, 20252′ hydroxy group
Manisha choudhary
August 26, 2025Rna m 2′ oh group hota h jo rna ko unstable bnata h DNA k compare m alkaline condition (NaOH ) m 2′ oh group paas waale phosphate group p attack kr deta h jis s degradation ho jata h
Priyanka Meena
August 26, 20252′ hydroxyl group
Muskan Yadav
August 26, 2025presence of a 2′-hydroxyl (-OH) group which is absent in DNA.
Kanica Sunwalka
August 28, 20252’oh grp in rna make it more unstable and reactive
Nikita Saini
August 28, 2025Major difference between RNA and DNA is due the presence of 2’OH group in RNA
Shriyanshi verma
August 30, 2025Only RNA is hydrolyzed by NaOH due to the additional 2′-OH group so correct answer is c
Alec
August 30, 2025option c – The presence of 2′- hydroxyl group makes it highly sensitive in alkaline condition.
Asha Gurzzar
August 30, 2025Option c is correct presence of hydroxyl group on rna pentose sugar
Pratibha Jain
August 30, 2025Correct answer is option (c)
presence of a 2′-hydroxyl (-OH) group
anjani sharma
August 30, 2025Presence of a 2′-hydroxyl (-OH) group which is absent in DNA.
Anisha jakhar
August 31, 2025Option c
Shobha Kanwar
August 31, 2025Ans (c) Presence of 2 hydroxyl gp in RNA makes it highly sensitive in alkaline condition.
Varsha Tatla
September 1, 2025+nce of 2’oh hydroxyl group
Deepika sheoran
November 15, 2025Option C is correct