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Lecture29 (Brandl6).docx

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Biochemistry 2280A
Eric Ball

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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 10  Equal to 3 X 10  That is there are a lot of molecules of template DNA in a sequencing reaction.  There is NOT just one!! Restriction Enzymes:  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 overhanging ends.  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 st  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 th  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 million  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  These include: o Temperature o pH o Salt concentration  They have enzyme kinetics DNA Ligase:  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  The activity
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