1. Electrophoresis & RFLPs
Advanced Techniques
Electrophoresis & RFLPs
Modified from: Kim Foglia, Explore Biology

2. Gel Electrophoresis Separation of DNA fragments by size
DNA is negatively charged
moves toward + charge in electrical field
agarose gel
“swimming through Jello”
smaller fragments move faster
cut DNA with restriction enzymes

3. Gel Electrophoresis

4. Gel Electrophoresis

6. Measuring fragment size
compare bands to a known “standard”
usually lambda phage virus cut with HindIII
nice range of sizes with a distinct pattern

7. RFLP Restriction Fragment Length Polymorphism
differences in DNA between individuals
change in DNA sequence affects restriction enzyme “cut” site
will create different band pattern

8. Polymorphisms in populations
Differences between individuals at the DNA level

9. RFLP 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”
“standard”
semen sample from rapist
blood sample from suspect
“standard”

10. RFLP 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
blood sample from victim 1
blood sample from victim 2
“standard”

11. Modified from: Kim Foglia, Explore Biology
Any Questions??
Modified from: Kim Foglia, Explore Biology

12. RFLP 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
blood sample from victim 1
blood sample from victim 2
“standard”

13. Advanced Techniques Part 2
Southern Blot, PCR, Sequencing, Human Genome Project
Modified from: Kim Foglia, Explore Biology

14. Southern Blot
Want to locate a sequence on a gel?

15. Southern blot Transfer DNA from gel to filter paper
hybridize filter paper with tagged probe
fragment with matching sequence “lights up”

16. Hybridization in Southern Blotting
Use radioactive probe to locate gene on filter paper
go back to gel & cut out piece of DNA you want to collect

17. Polymerase Chain Reaction (PCR)
What if you have too little DNA to work with?
PCR is a method for making many copies of a specific segment of DNA
~only need 1 cell of DNA to start
copying DNA without bacteria or plasmids!

18. PCR process It’s copying DNA in a test tube! What do you need?
template strand
DNA polymerase enzyme
nucleotides
primer
Thermocycler

19. What does 90°C do to our DNA polymerase?
PCR process
What do you need to do?
in tube: DNA, enzyme, primer, nucleotides
heat (90°C) DNA to separate strands (denature)
cool to hybridize (anneal) & build DNA (extension)
What does 90°C do to our DNA polymerase?

20. PCR primers The primers are critical!
need to know a bit of sequence to make proper primers
primers bracket target sequence
start with long piece of DNA & copy a specified shorter segment
primers define section of DNA to be cloned
PCR is an incredibly versatile technique:
An important use of PCR now is to “pull out” a piece of DNA sequence, like a gene, from a larger collection of DNA, like the whole cellular genome. You don’t have to go through the process of restriction digest anymore to cut the gene out of the cellular DNA. You can just define the gene with “flanking” primers and get a lot of copies in 40 minutes through PCR.
Note: You can also add in a restriction site to the copies of the gene (if one doesn’t exist) by adding them at the end of the original primers.
20-30 cycles
3 steps/cycle
30 sec/step

21. The polymerase problem
PCR cycles
3 steps/cycle
30 sec/step
Heat DNA to denature it
90°C destroys DNA polymerase
have to add new enzyme every cycle
almost impractical!
Need enzyme that can withstand 90°C…
Taq polymerase
from hot springs bacteria
Thermus aquaticus
Taq = Thermus aquaticus (an Archaebactera)
Highly thermostable – withstands temperatures up to 95°C for more than 40min.
BTW, Taq is patented by Roche and is very expensive. Its usually the largest consumable expense in a genomics lab. I’ve heard stories of blackmarket Taq clones, so scientists could grow up their own bacteria to produce Taq in the lab. It’s like pirated software -- pirated genes!
play DNAi movie

22. Kary Mullis 1985 | 1993 development of PCR technique
a copying machine for DNA
In 1985, Kary Mullis invented a process he called PCR, which solved a core problem in genetics: How to make copies of a strand of DNA you are interested in.
The existing methods were slow, expensive & imprecise. PCR turns the job over to the very biomolecules that nature uses for copying DNA: two "primers" that flag the beginning & end of the DNA stretch to be copied; DNA polymerase that walks along the segment of DNA, reading its code & assembling a copy; and a pile of DNA building blocks that the polymerase needs to make that copy.
As he wrote later in Scientific American:
"Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents and a source of heat. The DNA sample that one wishes to copy can be pure, or it can be a minute part of an extremely complex mixture of biological materials. The DNA may come from a hospital tissue specimen, from a single human hair, from a drop of dried blood at the scene of a crime, from the tissues of a mummified brain or from a 40,000-year-old wooly mammoth frozen in a glacier."

23. DNA Sequencing Sanger method determine the base sequence of DNA
dideoxynucleotides
ddATP, ddGTP, ddTTP, ddCTP
missing O for bonding of next nucleotide
terminates chain

24. DNA Sequencing Sanger method1
Sanger method
synthesize complementary DNA strand in vitro
in each tube:
“normal” N-bases
dideoxy N-bases
ddA, ddC, ddG, ddT
DNA polymerase
primer
buffers & salt 2 3 4 2

25. Reading the sequence
Load gel with sequences from ddA, ddT, ddC, ddG in separate lanes
read lanes manually & carefully
polyacrylamide gel

26. Fred Sanger 1978 | 1980 This was his 2nd Nobel Prize!!
1st was in 1958 for the structure of insulin

27. Advancements to sequencing
Fluorescent tagging
no more radioactivity
all 4 bases in 1 lane
each base a different color
Automated reading

28. Advancements to sequencing
Fluorescent tagging sequence data
Computer read & analyzed

29. Advancements to sequencing
Capillary tube electrophoresis
no more pouring gels
higher capacity & faster
Applied Biosystems, Inc (ABI) built an industry on these machines
384 lanes

30. Big labs! economy of scale PUBLIC Joint Genome Institute (DOE) MIT
Washington University of St. Louis
Baylor College of Medicine
Sanger Center (UK)
PRIVATE
Celera Genomics
Celera: Rockville, MD & San Francisco, CA
Baylor: Houston TX

31. Automated Sequencing machines
Really BIG labs!

32. Human Genome Project U.S government project Celera Genomics
begun in 1990
estimated to be a 15 year project
DOE & NIH
initiated by Jim Watson
led by Francis Collins
goal was to sequence entire human genome
3 billion base pairs
Celera Genomics
Craig Venter challenged gov’t
would do it faster, cheaper
private company
build the technology groundwork
improve sequencing methods
build clones
build better data management systems (computer tools to find overlaps)
better, cheaper, faster!
painstaking sequencing work
1998
Celera genomics challenge
2000
rough draft of human genome (90% sequence, 99% accurate)
2001
1st draft of human genome
2003
“finished” sequence of human genome
can’t sequence telomeres & centromeres

33. Different approaches “map-based method” “shotgun method” gov’t method
Craig Venter’s method
Cut DNA segment into fragments, arrange based on overlapping nucleotide sequences, and clone fragments.
2. Cut and clone into smaller fragments.
1. Cut DNA entire chromosome into small fragments and clone.
2. Sequence each segment & arrange based on overlapping nucleotide sequences.
3. Assemble DNA sequence
using overlapping sequences.

34. Human Genome Project
On June 26, 2001, HGP published the “working draft” of the DNA sequence of the human genome.
Historic Event!
blueprint of a human
the potential to change science & medicine

35. Sequence of 46 Human Chromosomes
3G of data
3 billion base pairs

36. Raw genome data

37. GenBank Database of genetic sequences gathered from research
Publicly available!

38. Organizing the data

39. Maps of human genes… Where the genes are…
mapping genes & their mutant alleles

40. And we didn’t stop there…
The main imperative for a primate genome project is to find out why humans develop certain diseases that other primates are immune to — like HIV.

41. The Progress # of DNA base pairs (billions) in GenBank
122+ bacterial genomes
first metazoan complete (flatworm)
first eukaryote complete (yeast)
17 eukaryotic genomes complete or near completion including Homo sapiens, mouse and fruit fly
First 2 bacterial genomes complete
In the last 5 years or so the amount of data has grown exponentially, including the growth of online databases and resources. In this slide we have the growth of the total number of base pairs and the total number of genomes completed since the beginning of the Human Genome Project. As you can see, the sheer number and growth of this resource has been impressive—and daunting—in the last 5 years.
Few scientists are aware of, or make full use of, all the open-source and public resources available to them through the internet. The Annual Nucleic Acids Research Database issue listing contained 548 databases this year!!
And, as this quote mentions, only half of those who use the databases are familiar with their tools. This Wellcome Trust study also made it clear that many people become users of a database after being told about it by colleagues.
“Despite the large amount of publicity surrounding the
Human Genome Project, a recent survey conducted on behalf
of the Wellcome Trust indicates that only half of biomedical
researchers using genome databases are familiar with the
tools that can be used to actually access the data.
In “The Molecular Biology Database Collection: 2003 update”
by Andreas D. Baxevanis in the Jan 1, 2003 NAR database issue.
# of DNA base pairs
(billions)
in GenBank
Official “15 year”
Human Genome Project:
Data from NCBI and TIGR
( and )

42. How does the human genome stack up?
Organism
Genome Size (bases)
Estimated Genes
Human (Homo sapiens)
3 billion
30,000
Laboratory mouse (M. musculus)
2.6 billion
Mustard weed (A. thaliana)
100 million
25,000
Roundworm (C. elegans)
97 million
19,000
Fruit fly (D. melanogaster)
137 million
13,000
Yeast (S. cerevisiae)
12.1 million
6,000
Bacterium (E. coli)
4.6 million
3,200
Human Immunodeficiency Virus (HIV)
9700 9

43. What have we found?
When you go looking…

44. …you will certainly find something!

45. Modified from: Kim Foglia, Explore Biology
Any Questions??
Modified from: Kim Foglia, Explore Biology

46. Modified from: Kim Foglia, Explore Biology
Advanced Techniques
Microarrays
Modified from: Kim Foglia, Explore Biology

47. Where do we go next…. DNA RNA protein trait
When a gene is turned on, it creates a trait
want to know what gene is being expressed
extract mRNA from cells
mRNA = active genes
How do you match mRNA back to DNA in cells???

48. slide with spots of DNA
each spot = 1 gene
Microarrays
Create a slide with a sample of each gene from the organism
each spot is one gene
Convert mRNA  labeled cDNA
mRNA  cDNA
mRNA from cells
reverse transcriptase

49. Microarrays Labeled cDNA hybridizes with DNA on slide
slide with spots of DNA
each spot = 1 gene
Microarrays
Labeled cDNA hybridizes with DNA on slide
each yellow spot = gene matched to mRNA
each yellow spot = expressed gene
cDNA matched to genomic DNA
mRNA  cDNA
Developed by Pat Brown at Stanford in late 1980s
Realized quickly he needed an automated system: robot spotter
Designed spotter & put plans on Internet for benefit of scientific community.

50. Application of Microarrays
2-color fluorescent tagging
Comparing treatments or conditions = Measuring change in gene expression
sick vs. healthy; cancer vs. normal cells
before vs. after treatment with drug
different stages in development
Color coding: label each condition with different color
red = gene expression in one sample
green = gene expression in other sample
yellow = gene expression in both samples
black = no or low expression in both
It’s all about comparisons!
Powerful research tool.

51. “DNA Chip” Patented microarray technology from Affymetrix
automated DNA synthesis of genes of interest on chip
chips are more consistent
smaller spots/more spots per chip
can buy specific chips
human chip
mouse chip
etc.
chip = $1000
machine to read chip = $150,000

52. Biotechnology today: Applications
Application of DNA technologies
basic biological research
medical diagnostics
medical treatment (gene therapy)
pharmaceutical production
forensics
environmental cleanup
agricultural applications
…and then there’s the ethics issues!

53. Application of recombinant DNA
Combining sequences of DNA from 2 different sources into 1 DNA molecule
often from different species
human insulin gene in E. coli (humulin)
frost resistant gene from Arctic fish in strawberries
“Roundup-ready” bacterial gene in soybeans
BT bacterial gene in corn
jellyfish glow gene in Zebra “Glofish”
In 1978, scientists at the Biotechnology Company, Genentech, cloned the gene for Human Insulin. Genentech licensed the human insulin technology to Eli Lilly, where it was named "Humulin" or Recombinant Human Insulin. In 1982, human insulin became the first recombinant DNA drug approved by FDA. Today, Humulin is made in Indianapolis in gigantic fermentation vats, 4 stories high and filled with bacteria!!! These fermentation vats operate 24 hours a day, year round. The human insulin protein made by the E. coli bacteria is collected from the vats, purified, and packaged for use by patients with diabetes.

54. Human Cloning
Human cloning is very controversial & not the main goal of biotechnology

55. Modified from: Kim Foglia, Explore Biology
Any Questions??
Modified from: Kim Foglia, Explore Biology

56. What next?
After you have cloned & amplified DNA (genes), you can then tackle more interesting questions
how does gene differ from person to person?
…or species to species
is a certain allele associated with a hereditary disorder
in which cells is gene expressed?
where is gene in genome?