1. DNA TECNOLOGY
AP Biology

2. MANIPULATE DNA to…
Create‘enhanced’ and novel organisms (GMO’s) via DNA recombination
Deduce phylogeny of species &
variation between genes via
genome sequencing
Identification of individuals
(criminals, dads) & allele
variations via DNA
fingerprinting

3. ISOLATE, then AMPLIFY, the DNA
Isolate the gene of interest, then replicate, using polymerase chain reaction - PCR
Nucleotides + primers + thermal cycling + a few hours = many, many copies
Requires the heat-stable Taq polymerase, originally isolated from the hot springs bacterium Thermus aquaticus (Mullis, 1982)
Kary Mullis, 1993 Nobel Prize in Chemistry

4. CUT DNA with RESTRICTION ENZYMES
Creates fragments of different lengths
aka ‘RFLP’s’: Restriction Fragment Length Polymorphisms
1.2 kb
0.2 kb
2 fragments
CCTGAGG……….. CCTGAGG…CCTGAGG
MstII
1.4 kb
1 fragment
CCTGAGG…………CCTGTGG…CCTGAGG

5. Restriction Enzymes • Used by bacteria to cut up foreign DNA
• Each recognizes a
specific DNA sequence
• Many are pallindromes,
read same in both
directions
makes blunt cuts
makes staggered cuts

6. Sort RFLP’s via gel ELECTROPHORESIS
aka DNA Fingerprinting

7. Measure small amounts of DNA precisely with a MICROPIPETTE
measures microliters (µL)
1000 µL = 1 mL
always use with a disposable tip!

8. Blunt cut DNA fragments are loaded into the wells of an
agarose gel

9. Electric current runs through
the gel
Negatively charged (PO4-)
DNA fragments migrate towards the positive electrode

10. • Fragments are thus sorted by size
• Short RFLPs (DNA fragments) diffuse through the gel more easily and quickly than longer fragments
• Fragments are thus sorted by size
Longest fragment
Shortest fragment

11. different restriction enzymes
Same DNA,
different restriction enzymes
Fragment
size in nt

12. same restriction enzyme
Different DNA,
same restriction enzyme
standard marker
(known reference) )
Who’s a match?

13. APPLICATION: Diagnose genetic disease
cctcagg cctcagg cctcagg
cctcagg cctctgg cctcagg
no cut site
•In sickle cell, the mutant form of the gene loses a recognition site
for a restriction enzyme
• creates one longer fragment when there should be two.

14. APPLICATION: Identify individuals
• Unlike genes, non-coding (nc) DNA differs greatly from person to person
• Use the ncDNA to identify people
gene
variable ncDNA sequence
attagctagcgtagcgggcatatatatggctaaattgcaccc
attagctagcgtagcgtccaatatatatatatatatatccgca
person 1
same different
person 2

15. ncDNA contains STRs
• Short, Tandem, Repeat sequences
• Numbers of repeats for any one location in the ncDNA is variable in a population
gggcatatatatggctaaattgcaccc gtccaatatatatatatatatatccgca
4 ‘at’ repeats:
9 ‘at’ repeats:

16. EXAMPLE • ncDNA (D7S280) of chromosome 7 includes the STR gata
• present in 6-20 repeats in the population
1 aatttttgta ttttttttag agacggggtt tcaccatgtt ggtcaggctg actatggagt
61 tattttaagg ttaatatata taaagggtat gatagaacac ttgtcatagt ttagaacgaa
121 ctaac gatag atagatagat agatagatag atagatagat agatagatag atagatagat
181 agata gtttt tttttatctc actaaatagt ctatagtaaa catttaatta ccaatatttg
241 gtgcaattct gtcaatgagg ataaatgtgg aatcgttata attcttaaga atatatattc
301 cctctgagtt tttgatacct cagattttaa ggcc
This STR is found on chromosome 7. The end of the sequence is actually …gata gaca gatt gata not sure whyt hat counts!

17. The FBI uses CODIS • A national DNA index system (1990)
• A database of convicted offender profiles for solving crimes for which there are no suspects.
• Process:
> Identify by gender, XX vs XY
(eliminates half the population)
> Use STR’s at 13 loci in the
genome to compare DNA samples
via gel electrophoresis
This STR is found on chromosome 7. The end of the sequence is actually …gata gaca gatt gata not sure whyt hat counts!

18. Possible, but not probable, for two people to have the same # of STR repeats at multiple loci
Probability of a person who is homozygous for the five highlighted ncDNA : 1 in 2.9 billion
(D15S1035 frequency2) x (each of the others2)

19. RELATED READING (links on Heredity webpage)
The Innocence Project exonerating wrongfully convicted individuals through DNA testing
The False Promise of DNA testing (The Atlantic) sloppy lab work
JUSTICE for ALL?
2004 Justice for All act: Grants any federal inmate the right to petition a federal court for DNA testing to support a claim of innocence
3 states, however, do not allow such a petition
2009: Supreme Court decided there's no constitutional right to DNA testing; the states and federal government can decide when testing should be allowed.
Nebraska Attorney General Jon Bruning hailed the decision as necessary to protect the states from unwarranted expense

20. WILDLIFE, TOO forest savannah Loxodonta africana
genetic differences indicate common ancestor several mya
Same species? Clear cranial, tusk morphology, genetic differences; Yet hybridize successfully
HHMI Video: Savannah elephant census
HHMI Click and Learn: Ivory poaching