September 5

1
September 5

• GATTACA
• Some comments about the quiz, practice
• A brief review
• Today-Methods
• DNA isolation and manipulation
• DNA sequencing
• PCR
• Sequencing genomes

Thursday finish methods, quiz second half of
class
2
The science in GATTACA

• Can you get a DNA sequence from one hair?
• How long would it take to get a DNA sequence of
  an individual?
• How much would it cost?
• How many pages would it fill?

3

• The latest NASA probe to Mars brings back some
  samples of living material that seems able to
  reproduce in the lab. These samples are of great
  interest because they may have evolved
  independently from life on earth. Two of these
  samples appear to be variants of the same
  species. Your goal is to figure out how they
  transmit their genetic material, so you take
  homogenates from one strain and are able to
  transform the second strain so that it more
  closely resembles the first strain.
• 1) How would you most easily identify the genetic
  material in this organism? (20 points)

4

• 1) Suppose only a tiny amount of the material you
  identify as the genetic material is necessary.
  How could you rule out contamination by some
  other material? (5 points extra credit)

5
DNA REPLICATION

• A lot of events are taking place at a replication
  origin-see if you can draw one and explain
  everything.
• Cellular DNA replication is the basis for DNA
  sequencing and PCR (which are easier)

6
Draw a replication origin (half)

• Leading and lagging strand, indicating direction
  of replication
• Where is the helicase? Topo I, Topo II?
• Where is the newest 50 bp of synthesized DNA?
• DNA primase? DNA ligase?
• Error mechanisms

7
DNA polymerase Free 3OH of a paired
base Synthesis is 5 to 3, the new nucleotide
is added to the 3OH
8
(No Transcript)
9
Here is half of an origin. Draw the other
half, Connecting and labeling 5 and 3 ends of
all strands
10
(No Transcript)
11
A couple of things to think about

• Complexity of DNA vs. proteins
• How can DNA code?
• What is the frequency of a given sequence?
• 6 base pair restriction site
• 20 base pair oligonucleotide

12
What is the frequency of a given 20mer?
13
What is the frequency of a given 20mer?

• (1/4)20
• 1/1,099,511,627,776

14
Working with DNA in the pre-PCR world

• DNA isolation
• Cutting and pasting DNA
• Cloning into libraries
• Genomic
• cDNA
• Using a probe to identify the sequence of
  interest

15
Isolating DNA

• Grind cells, remove protein by adding SDS,
  heating, extraction with organic solvent
• DNA precipitates well in 70 ETOH, 0.1M NaCl
• For PCR applications, heating may be all that is
  necessary

16
Visualizing DNA

• Fragments derived from plasmids or small pieces
  of DNA
• Genomes

17
Breaking DNA into pieces Shearing Or using
enzymes
18
Cloning pieces of DNA Making libraries
19
(No Transcript)
20
Visualizing DNA Agarose gels EtBr UV What
would DNA from a 6kb plasmid with 3 sites for a
restriction enzyme look like? A whole
organism, digested with a restriction enzyme and
run out on a gel look like?
21
Studying specific pieces of DNA
Fragments Libraries-where you get fragments of
interest from
22
(No Transcript)
23
(No Transcript)
24
Whats the difference between this and the
previous slide?
25
(No Transcript)
26
cDNA Library- A DNA copy of the mRNAs or messages
27
(No Transcript)
28
(No Transcript)
29
Now you have a library-so what!
30
Now you have a library-so what!

• Use to isolate a bigger piece around a gene
• Use to isolate a genomic clone
• Use to isolate a cDNA
• Use to isolate the gene in a different species

31
(No Transcript)
32
Tm

• This is an equation for the equilibrium between
  two strands of DNA
• Tm81.5 deg C .

33
Tm

• This is an equation for the equilibrium between
  two strands of DNA
• Tm81.5 deg C 16.6 log10M Na 0.41 () (GC)
  - 500/n - P - F
• nlength in nucleotides
• P mismatch
• F formamide
• AT Tm, 50 of base pairs are annealed. Usually
  you do your hybridizations at a lower temp than
  Tm.

34
(No Transcript)
35
Making probes

• Radioactive label
• Visual label (like digoxygenin)

36
(No Transcript)
37
(No Transcript)
38
Screening is done with membranes

• Screening a library-filter lifts
• Screening a gel-Southern blot

39
(No Transcript)
40
(No Transcript)
41
Hybridization can also be performed on whole
chromosomes
42
In situ hybridization on A) zebrafish brain B)
within nucleolus of a pea cell, white spots are
transcription of rDNA genes
43
DNA sequencing

• Remember, for DNA sequencing, you
• need a template, some DNA that you have lots of
  where you know the sequence of part of it so you
  can use an oligonucleotide primer

44
(No Transcript)
45
(No Transcript)
46
(No Transcript)
47
(No Transcript)
48
Read this gel 5 to 3 Lanes are GATC, from left
to right
49
Read this gel 5 to 3 Lanes are GATC, from left
to right
5TATAAACTGGACAACCAGTTCGAG3
50
Dye terminator technology
51
(No Transcript)
52
(No Transcript)
53
DNA sequencing-what do you need?
54
DNA sequencing-what do you need?
Template One primer DNA polymerase dNTPs ddNTPs Ei
ther labeled dNTP or dye terminators
55
The Polymerase Chain Reaction

• PCR
• Oligos, TM
• Thermostable polymerase

56
(No Transcript)
57
(No Transcript)
58
(No Transcript)
59
(No Transcript)
60
RT-PCR
61
(No Transcript)
62
(No Transcript)
63
(No Transcript)
64
(No Transcript)
65
So what are all of these methods good for?
66
PCR-what do you need?

• Template
• 2 primers
• DNA polymerase
• dNTPs

67
Recombinant proteins
68
Value added, or transgenic plants or animals
69
(No Transcript)
70
Information for basic biology to help understand
genetic diseases, infectious diseases
71
DNA sequencing of genes-what it tells us
72
With a DNA sequence, we can compare sequences of
DNA and proteins with all organisms whose
sequences are in the database
73
We can test under what Conditions genes are
expressed Microarrays
74
(No Transcript)
75
Genome sequencing

• How it is done
• Why we are sequencing so many genomes
• What it tells us
• What we can use the information for

76
Model systems

• We can inactivate single genes and ask what the
  function of that gene is.

77
(No Transcript)
78
(No Transcript)