This will be for a second step. Right now, you are saying that your file contains only /ACGT/i but that your validation procedure fails. Many of us think that it is likely that your file contains at least one line feed or carriage return character or a combination of both. The important thing right now is to find out what are the hidden characters that lead your validation subroutine to fail. Once you know that, you can modify your original program or your regex to take the findings into account.