Brandl - Lecture 6 Notes
It is important to recognize that most applications of Molecular biology use
chemical quantities of the DNA.
In a DNA sequencing reaction you generally add ~100 ng of DNA. Assuming the
DNA is 3000bp-14pproximately how many moles of template are added?
o 5X10 moles
o 1 mole of a DNA of 3000bp has a mass of:
3000 X 660g/bp ~2 X 10 grams in one mole.
100 ng has 5 X 10 -14moles
Equal to 3 X 10
That is there are a lot of molecules of template DNA in a
There is NOT just one!!
Restriction enzymes are usually type II enzymes, which show specific
recognition and specific cleavage of DNA.
There are about 300 Type II restriction enzymes now known.
They are site-specific DNA binding proteins, which in general recognize and cut
palindromic sequences most commonly of 4, 6 and 8 bases
Cleavage by EcoR1
It recognizes the sequences 5’GAATTC3’
It cuts after the G.
It is symmetric on both strands (leading and lagging)
After cleaving you get 5’ overhangs on either end (single stranded)
The 5’ end is what is overhanging
EcoR1 leaves sticky ends with 5’ overhangs
Cleavage by Sst1
It recognizes 5’GAGCTC3’ (palindromic sequence)
It cuts between the T and the C, and symmetrically on the other strand.
There is a 3’ overhang this time
SsT1 cleaves to leave sticky ends with 3’ overhangs
Cleavage by Sma1
It recognizes the palindromic sequence 5’CCCGGG3’
It cuts between the C and the G
Sma1 cleaves to blunt ends
Some restriction enzymes cut identical sequences, but leave different
BamH1- cuts 5’GGATCC3’ - it cuts between the two G’s & gives 5’ overhangs
NIAIV- cuts 5’GGATCC3’ - cuts and gives blunt ends
BamHI- cuts 5’AGATCT3’ - cuts between the A and the G & leaves identical 5’
overhangs as BamHI What is the probability that any random 4 bp sequences is a HaeIII site (GGCC)?
1 in 256
Probability that the 1 pondtion is G is 1 in 4
The probability that the 2 position is G is 1 in 4
The probability that the 3 position is C is 1 in 4
The probability that the 4 position is C is 1 in 4
¼ X ¼ X ¼ X 1/4 = 1/256
Given that the probability of any 6bp sequence being a HincII site (GTPyPuAC) is
~1/1000, approximately how many Hincll sites are found within the human genome
(3 billion bp)?
3 X 10 /10 = 3 X 10 6
Restriction Enzymes are Enzymes:
Like any other enzyme, EACH restriction enzyme has preferred conditions in
which it functions
o Salt concentration
They have enzyme kinetics
While restriction enzymes act as scissors, the enzyme DNA ligase acts as glue
If there is a 5’ phosphate, it will reseal compatible sticky ends
Ligase will also reseal ligate blunt ends, but this will be less efficient
The reaction requires an energy source (hydrolyzes ATP)
The enzyme is usually isolate from the T4 bacteriophage