1. DESKTOP RFLP ANALYSIS Brookings Biology Kelly Riedell
SOME SLIDES MODIFIED FROM KIM FOGLIA
DESKTOP RFLP ANALYSIS
Brookings Biology
Kelly Riedell

2. Big Idea 3: Living systems store, retrieve, transmit and respond to information essential to life processes. Enduring understanding 3.A: Heritable information provides for continuity of life. Essential knowledge 3.A.1: DNA, and in some cases RNA, is the primary source of heritable information e. Genetic engineering techniques can manipulate the heritable information of DNA and, in special cases, RNA. To foster student understanding of this concept, instructors can choose an illustrative example such as: • Electrophoresis • Plasmid-based transformation • Restriction enzyme analysis of DNA • Polymerase Chain Reaction (PCR)
SP 6: The student can work with scientific explanations and theories. 6.1 The student can make claims and predictions about natural phenomena based on scientific theories and models.
6.2 The student can construct explanations of phenomena based on evidence produced through scientific practices

5. RFLPs Restriction Fragment Length Polymorphism
FROM KIM FOGLIA
RFLPs
Restriction Fragment Length Polymorphism
differences in DNA between individuals
Alec Jeffries 1984
change in DNA sequence affects restriction enzyme “cut” site
creates different fragment sizes & different band pattern

6. Differences at the DNA level
FROM KIM FOGLIA
Differences at the DNA level
Why is each person’s DNA pattern different?
sections of “junk” DNA
doesn’t code for proteins
made up of repeated patterns
CAT, GCC, and others
each person may have different number of repeats
many sites on our 23 chromosomes with different repeat patterns
GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTT
CGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA
GCTTGTAACGGCATCATCATCATCATCATCCGGCCTACGCTT
CGAACATTGCCGTAGTAGTAGTAGTAGTAGGCCGGATGCGAA

8. Comparing cut up DNA How do we compare DNA fragments?
FROM KIM FOGLIA
Comparing cut up DNA
How do we compare DNA fragments?
separate fragments by size
How do we separate DNA fragments?
run it through a gelatin
agarose
made from algae
gel electrophoresis

9. Gel electrophoresis – +
FROM KIM FOGLIA
Gel electrophoresis
A method of separating DNA in a gelatin-like material using an electrical field
DNA is negatively charged
when it’s in an electrical field it moves toward the positive side
DNA         – +
“swimming through Jello”

10. Gel electrophoresis – + DNA moves in an electrical field…
FROM KIM FOGLIA
Gel electrophoresis
DNA moves in an electrical field…
so how does that help you compare DNA fragments?
size of DNA fragment affects how far it travels
small pieces travel farther
large pieces travel slower & lag behind
DNA        – +
“swimming through Jello”

11. Running a gel 1 2 3 fragments of DNA separate out based on size
FROM KIM FOGLIA
Running a gel
fragments of DNA separate out based on size
cut DNA with restriction enzymes 1 2 3
Stain DNA
ethidium bromide binds to DNA
fluoresces under UV light

13. FROM KIM FOGLIA http://www. explorebiology
Uses: Forensics
Comparing DNA sample from crime scene with suspects & victim
suspects
crime scene sample S1 S2 S3 V –
DNA  +

14. FROM KIM FOGLIA http://www. explorebiology
DNA fingerprints
Comparing blood samples on defendant’s clothing to determine if it belongs to victim
DNA fingerprinting
comparing DNA banding pattern between different individuals
~unique patterns

15. RFLP / electrophoresis use in forensics
FROM KIM FOGLIA
RFLP / electrophoresis use in forensics
1st case successfully using DNA evidence
1987 rape case convicting Tommie Lee Andrews
“standard”
semen sample from rapist
blood sample from suspect
“standard”
How can you compare DNA from blood & from semen? RBC?
“standard”
semen sample from rapist
blood sample from suspect
“standard”

16. Electrophoresis use in forensics
FROM KIM FOGLIA
Electrophoresis use in forensics
Evidence from murder trial
Do you think suspect is guilty?
blood sample 1 from crime scene
blood sample 2 from crime scene
blood sample 3 from crime scene
“standard”
blood sample from suspect
OJ Simpson
blood sample from victim 1
N Brown
blood sample from victim 2
R Goldman
“standard”

17. Uses: Evolutionary relationships
FROM KIM FOGLIA
Uses: Evolutionary relationships
Comparing DNA samples from different organisms to measure evolutionary relationships
turtle
snake
rat
squirrel
fruitfly – 1 3 2 4 5 1 2 3 4 5
DNA  +

18. Uses: Medical diagnostic
FROM KIM FOGLIA
Uses: Medical diagnostic
Comparing normal allele to disease allele
chromosome with normal allele 1
chromosome with disease-causing allele 2
allele 1
allele 2 –
DNA 
Example: test for Huntington’s disease +

19. Uses: Paternity – + Who’s the father? 
FROM KIM FOGLIA
Uses: Paternity
Who’s the father? –
Mom F1 F2
child
DNA  +

20. LAY OUT 2 YARN PIECES ON YOUR DESK DON’T STRETCH
LAY OUT 2 YARN PIECES ON YOUR DESK DON’T STRETCH ! TRIM YOUR PURPLE & GEEN YARN PIECES SO THEY ARE THE SAME LENGTH- 50 cm
Image from :

21. RESTRICTION ENDONUCLEASES
GEL #1 Use the EcoR1 restriction enzyme provided to: Individual #1 (PURPLE yarn) – Make cuts at 10 cm AND 32 cm
Individual #2 (GREEN yarn) Cuts at 10 cm, 15 cm, & 40cm

22. LAY OUT YOUR TAN YARN PIECE ON YOUR DESK DON’T STRETCH !
CUT PIECES THAT ARE: 10cm, 20cm, 30cm, and 40cm long
This will be your “LADDER” DNA of known size to compare against
Image from :

23. SET UP YOUR DESKTOP AS A GEL
Use sticky notes to LABEL THE and – poles
Use sticky notes to LABEL your lanes
Load your “gel” with
your “DNA” that has been cut with “restriction enzymes”
Complete RFLP analysis
and draw a picture of
the results
LADDER TAN
#1 PURPLE
#2 GREEN –
DNA 
Use sticky
notes
to mark distances 40 30 20 10 +

24. RESTRICTION ENDONUCLEASES
GEL #2: Use DIFFERENT “restriction enzymes” to cut the SAME DNA Cut one piece of yarn with EcoRI –Cuts at 10 cm AND 32 cm
Cut one piece of yarn with HindIII– Cuts at 22 cm
Cut one piece of yarn with BOTH EcoRI AND HindIII

25. SET UP YOUR DESKTOP AS A GEL
Set up your new gel as before
Load your “gel” with
your “DNA” that has been cut with “restriction enzymes”
Complete RFLP analysis
and draw a picture of the results
EcoR1 and
HindIII
LADDER TAN –
EcoR1
HindIII
DNA  40 30 20 10 +

26. EXPLAIN WHY DNA moves in an electric field toward the POSITIVE pole
EXPLAIN WHY DNA moves in an electric field toward the POSITIVE pole. EXPLAIN the relationship between FRAGMENT SIZE and DISTANCE MOVED on a gel.

27. Linus Pauling used RFLP analysis to show that the sickle cell mutation caused a change in the DNA code. He cut the gene from a person with normal hemoglobin and a person with sickle cell anemia with a restriction enzyme and compared them using RFLP analysis. Use what you know about the sickle cell mutation and restriction recognition sites to EXPLAIN how a mutation could change the NUMBER and LENGTHS OF FRAGMENTS on a gel .
SP 6: The student can work with scientific explanations and theories The student can construct explanations of phenomena based on evidence produced through scientific practices

28. http://image. slidesharecdn

29. http://image. slidesharecdn
MAKE A PREDICTION about what the gel pattern from a person who is heterozygous for the sickle cell allele might look like. HINT: Think about what the word HETEROZYGOUS means.
SP 6: The student can work with scientific explanations and theories. 6.1 The student can make claims and predictions about natural phenomena based on scientific theories and models.

30. Give at least FOUR (4) EXAMPLES of how DNA ANALYSIS can be used to compare DNA.