II Manipulating DNA A Isolating a specific DNA

(II) Manipulating DNA A. Isolating a specific DNA segment a. this would allow researchers to compare DNA samples b. it may allow researchers to transfer DNA from one organism to another

B. Cutting the DNA segment: Restriction endonucleases a. enzymes that cut ds. DNA at a specific recognition site b. these recognition sites are PALINDROMES which are sections of DNA that read the same 3’ 5’ in one way as they do 3’ 5’ the other way c. eg. GTGCAC CACGTG GAATTC C T T AA G AAGCTT TTCGAA

d. the restriction endonucleases often cut the DNA unevenly [as shown previously] and leave sticky ends which are short pieces of ss. DNA that will be able to H-bond to similar ss. DNA pieces e. generally a piece of DNA will have a recognition site for any one restriction enzyme every 5000 bp or so, spaced at random along the DNA [eg 2000, 9000, 1400, 16000 23000] or

f. the restriction enzymes are named after the bacteria that they are extracted from Esherichia coli strain R isolate 1 Eco. R 1 from Haemophilis influenza strain Rd, isolate 3 Hind III

C. Methylases a. These groups add methyl groups to the nitrogenous bases b. A methylated base will easily be recognized by a restriction enzyme & the DNA will NOT be bisected. c. This allows researchers to protect sections of DNA before using the restriction enzymes DNA Ligase d. This enzyme attaches ss. DNA complementary base pairs e. After, the sticky ends form phosphodiester bonds to hold it together

D. Separating the DNA Gel electrophoresis a. a procedure for separating the cut pieces of DNA to allow researchers to select the one they want

b. a gel is a jello-like substance made of starch, agarose or polyacrylamide

Loading samples on the gel

c. electrophoresis refers to separation based on electrical charge

d. DNA has a negative charge due to the phosphates OII – P – OII O-

e. smaller pieces of DNA will move further than large pieces of DNA – separation by size

Note: We do not see the different colours, just the pattern of different lengths The number of bands as well as the pattern may differ between samples

f. so that researchers can find the DNA on the gel – ethidium bromide is added this chemical fits into the twists of DNA without altering it and fluoresces under UV light

DNA with Ethidium Bromide

g. Many copies of DNA must be used in order to get enough of a sample to be visible.

h. DNA pieces can now be cut, separated & isolated from the gel

5. Using DNA fingerprinting [which does not involve fingerprints] a. This depends on the fact that some parts of our introns are highly variable. b. Since these introns do not code for a protein, this variability does no harm.

c. The DNA from any two people when cut with the correct endonucleases, will produce a different pattern of DNA segments. d. These segments can now be run on a gel and separated based on size. e. The unique pattern of these sections produced by this technique gives us a unique DNA fingerprint

f. Paternity tests the child’s DNA has to be able to be derived from the mother and the father mom baby dad 1 dad 2 plus mom can equal baby dad 1 plus mom does NOT equal baby

f. Criminal investigations eg. rape where the victim and the suspect are both sampled The specimen clearly excludes suspect 2 & 3, but things look bad for suspect 1

eg. murder where matching the suspect is the key both the suspect [defendant] and the victim are sampled the blood on the pants is the suspect’s the blood on the shirt is not the suspect’s the blood on the shirt is in fact the victim’s

show PCR animation