It does not and should not affect Southern Hybridization

Biotin normally should not affect the efficiency of hybridization like digoxigenin used in chemiluminesce labeling.

Thanks for the answers. But if biotin don't interfere the hybridization then why do companies sell two separate biotin and claim that the expensive one will not interfere the hybridization?

A number of modified protocols for photolabeling and chmiluminescent labeling are available.And there may be some variation in efficiency though the basic methodology is not changed much

I fully agree with Dr. Sivaramakrishnan. Note companies are commercial organizations........

For internal labeling, many commercial companies offer "Biotin-dT" (a biotin-modified thymidine residue that can be used internally in place of thymidine). Alternatively, there is the "Biotin-ON", which is used primarily for applications where the user doesn’t want to incorporate a thymine base into their oligonucleotide sequence, but wants to hybridize the oligo to a complementary target. The type of Biotin modification you use DOES affect hybridization! Especially if it is internally placed. Biotin-TEG is a commonly used Biotin mod because of its higher affinity for avidin. Note: The affinity of avidin for biotin is decreased when short spacer arms are used in biotinylated compounds due to steric hindrance, and since Biotin-TEG has a 15-atom spacer it is usually the mod of choice. However, internal Biotin-TEG modifications will disrupt hybridization due to the size of the TEG molecule. Biotin-dT should not affect hybridization, but could have a lower affinity to your avidin. In alignment, you should note that for Biotin-dT the Biotin is attached to the base via an aliphatic chain, so there is still the possibility of some steric hindrance during hybridization. If hybridization efficiency is paramount in your application then perhaps using a 5' or 3' end mod would better serve your purpose.

Thanks Jonas for your expert views! I just want to share the results of our experiment. We used the biotin -1A-dATP (19524-016; Invitrogen) in PCR to label the PCR product (~300 bp product size). used this PCR product to hybridized with nylon membrane cross linked with probe and few spots with PCR product itself (probe were about 20 mer). We used Biotin Chromogenic Detection Kit (K0661, K0662; Thermo Scientific) for colour matrix based detection. After hybridization, we got the blue spots only with PCR product labelled on nylon membrane hybridized with biotin labelled PCR product but we never got any colour spot with probes. Do you have any idea that what could be the possible reason for it. I was thinking, it could be because of biotin that interfering the hybridization due to steric hindrance. What do you think? It might be possible that we are getting positive result when ~300 bp long PCR product hybridized with biotin labelled 300 bp PCR product because of long product size that minimized the effect of biotin hindrance. Due to long size, in some places nucleotide hybridized and didn't washed out during stringent washing steps. Let me know your views. Thanks