| M W F; 10:10 - 11:00 pm |
Molecular Biology |
Douglas W. Smith |
| York 2722 |
BIMM 100 |
5254 Muir Biology Building |
| Fall, 2000 |
x42620; dsmith@ucsd.edu |
| BIMM100 | Syllabus
| Sections / Off Hrs | Grading
Policy | DNASYSTEM
|
| Lectures | Journal
Articles | Study Qs | Lab
Techniques | Exams |
Outline:
A. Physical Mapping using R.enzymes, R.sites, and R.frags
Physical Mapping: determination of actual physical position of markers and features on the molecules comprising Genomes, i.e. DNA molecules.
Discoveries associated with Recombinant DNA Technology were key to the development of methodologies to determine these positions.
Key discoveries or developments:
1. Type II Restriction and Modification Enzymes [Brown, Tech Notes 3.1]
2. Gel Electrophoresis DNA fragment analysis [Brown, Tech Notes 3.2]
3. DNA Sequencing methodologies [Brown, Chapter 4]
4. Polymerase Chain Reaction ... PCR ... [Brown, Tech Notes 2.2]
The first two discoveries permitted mapping of Restriction Sites (R.Sites) on DNA molecules.
R.sites are short DNA sequences recognized and cleaved by enzymes called type II Restriction Enzymes (R.enzymes). DNA so cleaved with one or more R.enzymes yields fragments of DNA called Restriction fragments (R.frags).
Such cleavage with subsequent use of Gel Electrophoresis to determine the sizes of the R.frags and their positions relative to each other permits mapping of the positions of these R.sites.
This yields a Restriction Map of the DNA which can be a chromosome or the entire Genome of the organism.
1. Type II Restriction Enzymes:
R.enzymes
... [Brown, Tech
Notes 3.1]
Small dimer proteins that are DNases
a. Types of DNases (deoxyribonucleases):
1. Exonucleases: DNases
that cleave DNA from their ends
Often show specificity: ssDNA vs dsDNA; 5' ends vs 3' ends
2. Endonucleases: DNases
that cleave DNA within the molecule
Specificity: ssDNA vs dsDNA; 5' vs 3' Phosphate product
For endonucleolytic cleavage of dsDNA:
Nickase: cleave one DNA strand
Chopase: cleave both DNA strands opposite (or nearly opposite)
each other
Type II Restriction Enzymes
are Endonucleases that
Recognize specific, usually 4-6 bp sequences in dsDNA: R.Sites
Recognition sequence is usually a Nucleotide Palindrome:
Sequence same 5' to 3' on both DNA strands
Cleave backbone of both strands at unique positions
Cleavage points are usually not directly opposite to each other
but close: Chopases
This feature yields Sticky Ends ... short regions of ss
DNA
Examples: ... [Brown, Tech Notes 3.1]
EcoRI ... G'AATTC ... 6 cutter ... cleavage between the
G and A in each strand
=> 4 bp sticky end: AATT
BamHI ... G'GATCC ... 6 cutter ... cleavage between the
two G's in each strand
=> 4 bp sticky end: GATC
NotI ... GC'GGCCGC ... 8 cutter ... cleavage between the
C and G indicated
=> 4 bp sticky end: GGCC
HaeIII ... GG'CC ... 4 cutter ... cleavage between the G
and C indicated
=> blunt ends produced, no overhangs
HhaI ... GCG'C ... 4 cutter ... cleavage between the G
and C indicated
=> 2 bp sticky end: GC, at 3' end of the Restriction fragment
AluI ...
AG'CT ... 4 cutter ...
cleavage between the G and C indicated
=> blunt ends produced, no overhangs ...
AluI is the R.enzyme which identified Alu fragments:
common example of Middle Repetitive DNA ...
Sau3A ... 'GATC ... 4 cutter ... cleavage 5' to the G as
indicated
=> 4 bp sticky end: GATC
Note: This is the same overhang as for BamHI
above ...
can thus "ligate" BamHI and Sau3A fragments together
!!!
b. Fragment average size: ... highly variable sizes ...
6 bp Recognition Seq =>
occurs once every 46 = 4096 bp ~ 4 kb
Note: 210 = 45 = 1024 ~ 1000 = 103
... a convenient number to remember ...
Size of bacterial gene: ~ 1000 bp ... hence, good chance of entire
gene on R.frag
Human DNA: genes much bigger
... Intro-Exon structure ... genome big: 3300 Mb
=> ~ 800,000 EcoRI fragments ... can not resolve on gels; too
many
use NotI ... R.frags ~ 65 kb
in size (convince yourself of this !!!)
=> ~ 50,000 human DNA frags ... still very large ...
c. Cognate Modification Enzymes:
Each Type II R.enzyme has a
cognate Modification Enzyme: DNA methylase
This enzyme recognizes the same nucleotide sequence as does
the R.enzyme
and methylates specifically one of the bases in each strand in
the nuc sequence.
Example: EcoRI methylase ... GAA*TTC ... methylates the inner A in both strands
Function: this methylation renders this R.site
resistant to cleavage by the R.enzyme
Hence, a cell encoding a Restriction Enzyme also encodes the Modification
Enzyme;
the latter will methylate the cell chromosomal DNA, thereby preventing
the R.enzyme from digesting the cellular DNA.
2. Gel Electrophoresis of DNA fragments:
Digestion of a well defined
DNA molecule, eg viral DNA, with a R.enzyme
yields a set of well-defined DNA Restriction Fragments ...
R.frags
Example: EcoRI cleaves Phage Lambda DNA (linear dsDNA, 49 kb) at 5 sites, yielding 6 fragments
BamHI cleaves Adenoviral DNA at 12 sites, Lambda DNA at 5 sites ...
The sizes of these R.frags
can be determined using Gel Electrophoresis
and the fragments can often be isolated from each other
Gel Electrophoresis: ... [Brown, Tech Notes 3.2]
When "loaded" as
a "band" at top of a gel matrix, and subjected to an
Electric
Field, DNA migrates through the gel at rate inversely proportional
to its size
... rough exponential dependence of migration rate with size ...
Thus, can
1. separate and purify individual
R.frags from a gel
2. can analyse for presence and size of R. frags
Use DNA Markers for
size determination ... avoid variation between gel runs
Methodology:
"Slab" gels used:
several DNA samples in adjacent "lanes" analyzed in
same gel
Agarose gels used for R.frags of size 500 bp or larger ... [Brown,
Tech Notes 3.2]
Polyacrylamide gels used for
smaller fragments, e.g. sequencing gels
... [Brown, Fig 4.1; Tech Notes 4.1]
"Pulsed gel" electrophoresis
used for very large DNA, eg entire chromosomes
... [Brown, Fig 3.5; Journal Article 1 - YAC paper]
Visualize the DNA: ... [Brown, Tech Notes 3.3]
1. Using radioactivity: place
X-ray film over gel, expose to decaying P32
2. fluorescence: Ethidium Bromide (EtBr), a dye which intercalates
between bases ...
... EtBr fluorescences
in visible when irradiated with UV light ... [Brown, Tech Notes
3.2]
3. Physical Map of Genome
Restriction Map as a Physical Map: ... [Brown, Fig 3.2,
3.3]
Given a large DNA molecule:
1. digest it with an R.enzyme,
measure sizes via Agarose Gels
2. repeat with additional R.enzymes
BUT only get sizes, don't know
which fragments are adjacent to each other
3. do DOUBLE digests with the same enzymes as above, 2 at a time
purify singly-digested R.frags
and digest with the 2nd R.enzyme
(can often avoid this, or purify only a few of the first R.frags...)
=> get Ordering of Both
Enzyme set of fragments ...
4. can also do PARTIAL DIGESTS
with single R.enzyme ...
This is particularly useful for END LABELED DNA ...
assay P32 added at 5' ends of DNA with polynucleotide
kinase [Brown, Tech Notes 4.2]
Can also use hybridization of a radioactive 'probe' ssDNA or oligonucleotide to one end or the other of the DNA in a Southern gel analysis of the partial digests ... see Journal Article 1, the YAC paper, Fig. 5
Physical Maps based on R.sites are now available for most "model"
organisms ...
e.g. Kohara map for E. coli ... Sau3A mapping of C.
elegans
... as well as ultimate Nucleotide Sequence for several bacteria,
archae, and yeast.
Level of "resolution" depends on 8 vs 6 vs 4 bp "cutter"
vs DNA sequence ...
B. Physical Mapping using Hybridization
Properties of conversion of dsDNA into ssDNA, and the reverse, can also be used for Physical Mapping and other purposes. These properties have to do with DNA Hybridization.
1. DNA Hybridization - Melting Curves
a. Denaturation: conversion of dsDNA to two strands of ssDNA
1. When the H bonds break that
join the 2 strands in dsDNA, they all tend to break simultaneously
... thus, the dsDNA "melts" into two strands
This is like a phase transition similar to the melting
of ice into water ...
2. Agents which "denature" DNA:
a. Increase the temperature
past the "melting" temperature: Tm ...
[Brown, Fig 2. 7]
b. Increase the pH to above about 11.3
c. Decrease the ionic strength to below 10(-5): repulsion of negative
charge.
Assay or Measurement of Denaturation / Renaturation: UV light Absorption
1. The bases (mainly Pyrimidines) absorb UV light (260 nm) that impinges vertically on the base.
2. Free bases absorb more than
ssDNA; ssDNA absorbs more than dsDNA due to this requirement of
vertical impingement of the UV light:
bases most exposed when free; least exposed when in dsDNA; only
partially exposed in ssDNA due to "stacking" of the
bases (hydrophobic interactions)
3. Can measure this Denaturation
via change in UV absorption.
This is an assay, or measurement method, for this process
4. The Tm is the "midpoint" in the melting reaction ... Tm is proportional to %(G+C), due to 3 H-bonds between C and G but only 2 H-bonds between A and T
5. The curve of Assay (here: UV absorption) vs Melting Agent (here: Temperature) is called a Melting Curve.
b. UV light Absorption:
1. The bases (mainly Pyrimidines) absorb UV light (260 nm) that impinges vertically on the base.
2. Free bases absorb more than
ssDNA; ssDNA absorbs more than dsDNA due to this requirement of
vertical impingement of the UV light:
bases most exposed when free; least exposed when in dsDNA; only
partially exposed in ssDNA due to "stacking" of the
bases (hydrophobic interactions)
c. Renaturation: joining of two DNA strands of complementary sequence to form dsDNA
1. Reverse of Denaturation ... but requires that the two DNA strands have complementary sequence, i.e. permit A joining to T, C joining to G, along the entire length of both DNA strands or chains.
2. Renaturation is also called Hybridization ... [Brown, Tech Notes 2.1]
3. Can also occur between DNA
and RNA, to form a DNA:RNA hybrid, or between two RNA strands
of complementary sequence.
2. DNA Labeling
DNA probes used in hybridization experiments must be labeled in order to assay their association with a ssDNA substrate in a hybridization experiment.
DNA labels are most often either radioactive labels or fluorescent labels.
a. Radioactive Labeling
Radioactive labels used are radioactive isotopes:
H3, C14, P32, or P33
The most often used isotopes are H3 and P32
Methods of radioactive labeling:
b. Fluorescent Labeling
Primarily, radioactive labeling suffers from the obvious problem that people are exposed to radioactivity. This requires specific and special environmental safeguards. Such safeguards do not however solve the problem of longterm isotopes: isotopes that take thousands or millions of years to decay away - how does one dispose of these? ... this is an unsolved issue ...
Second, radioactive labels are not simultaneously of high sensitivity and of high resolution:
High sensitivity: high signal to noise ratio ... decay from the label is high compared with cosmic rays and other background 'noise'
High resolution: ability to pinpoint precisely in the experiment the position of radioactive decay of the radioactive atom.
P32: high sensitivity, low resolution ... Beta decay, half life = 14.2 days (half of the atoms have decayed in two weeks), max energy of the beta particle (high energy electron): ~ 1.3 Mev ... this is high energy; will pass through one or more cm of human tissue ...
H3: low sensitivity, high resolution ... Beta decay, half life ~ 1100 years, max energy of the beta: ~ 0.01 Mev ... low energy (low sensitivity), doesn't go far (high resolution)
Other radioisotopes are in between ...
Fluorescent labels are by comparison of highsensitity and high resolution.
These are used routinely in automated DNA
sequencing reactions and now in DNA microarray experiments.
One can purchase fluorescent labeling nucleotide triphosphates
and substrates for oligonucleotide (probe) synthesis.
Fluorescent lables of different wavelengths, green and red particularly, are available and can be used simultaneously in a given experiment, e.g. in DNA microarray experiments
3. Fluorescence In Situ
Hybridization - FISH
... [Brown, Chapter
3.2]
FISH is used to detect the position of a marker on a chromosome via fluorescence. The marker is a fluorescent-labeled ssDNA probe of complementary sequence to that of the position on the chromosome.
Methodology - [Brown, Fig 3.7]
Metaphase chromosomes (or other DNA substrate) is immobilized
on a glass slide
The chromosomes or DNA are denatured, e.g. with formamide
The fluorescent-labeled probe is hybridized to the denatured DNA
The DNA is visualized via microscopy or other method and the position
of the hybrized fluorescent probe is visualized via its fluorescent
signal: assay
Example: fly DNA probed with centromeric sequences - [Brown, page XXX; very beginning of Part 1 of the book, just before Chapter 1]
C. Single R.frag Identification:
Southern Hybridization
... [Brown, Tech Notes 2.1]
Often use a short DNA oligonucleotide
Probe to partially purify
R.fragments for further cloning, or to identify a given R.fragment,
or other DNA fragment containing SPECIFIC DNA sequences.
One can do this via a combined Agarose Gel Electrophoresis with Hybridization against a radioactive Probe that has a complementary DNA sequence to the desired specific DNA sequences ...
Named for Ed Southern, famous English molecular biologist ...
Can also use Hybridization
of a Probe to a Gene in order to:
1. detect DNA spotted and denatured on filter paper: dot blots
... []
2. disrupted Lambda phage from a Lambda cDNA library on plates
... []
3. lysed cells on a plate with DNA transferred to a nitrocellulose
membrane ...
in situ Hybridization ... []
1. Methodology: ... [Brown, Tech Notes 2.1]
... Examples: ; several figures in Journal Article 1 on YACs
1. Run DNA fragments out on an Agarose Gel
2. Float Agarose Gel on an
Alkaline solution in a trough ... []
Alkali denatures the DNA, yielding ssDNA fragments in the Agarose
Gel
3. Place a Nitrocellulose Membrane on top of the Agarose Gel ... and then several layers of absorbant paper (towel paper often used ...) on top of the Nitrocellulose Membrane
4. Let capillary action transfer DNA to the Nitrocellulose Membrane during transfer of fluid from the trough to the absorbant paper
5. Dry Nitrocellulose Membrane and fix DNA to the Nitrocellulose Membrane: 80 C, under vacuum
6. Hybridize the Radioactive Probe to the denatured ssDNA on Nitrocelluose Membrane ... hybridization conditions in small liquid volume in a "Seal-a-Meal" sac often used ...
7. Dry Nitrocellulose Membrane and expose radioactivity to film
8. Develop and examine the film ...
Such gels can be used analytically to
provide final information:
1. identify whether DNA in a lane contains the probe sequences:
see hybridization
2. see how many such bands there are
3. determine their size from position in the gel
Southern gels can also be used preparatively
to purify DNA fragments:
1. cut the band out of the gel and elute the DNA from the gel
slice
Such partially purified fragments can then be cloned ...
2. Variants of Southern Gels
1. Northerns: same as Southerns but with RNA, e.g. mRNA, run out on the Agarose Gels
2. Westerns: proteins run out on a Polyacrylamide gel (PolyAcrylamide Gel Electrophoresis - PAGE; generally under protein denaturing conditions, e.g. use of detergent: SDS gels) ... assay presence of a given protein using an Antibody to the protein ... []
Names here are "take-offs" on Southern gels ... not named for scientists named Northern, etc
Other variants also exist, e.g. South-Westerns (run SDS protein gel; attempt to renature proteins in gel; transfer to Nitrocellulose as in Southerns; probe the Proteins so transferred with Radioactive Oligonucleotide probes ... assay DNA binding Proteins)
D. Genome Fragment Identification: DNA Microarrays
One of the profound technological advancements that is a product of this third revolution in Molecular Biology is that of DNA Microarray technology.
DNA Microarrays are similar to Southern Gels in that labeled oligonucleotide probes are used to identify specific DNA sequences from among many, e.g. an entire genome.
However:
1. Southerns (and Northerns)
are one gene at a time technology.
2. DNA Microarrays permit simultaneous assay of all
genes in a genome
DNA Microarrays thus can be thought of as "the Southerns
of Genome-based Molecular Biology"
1. Methodology
DNA Microarrays are 2D solid surfaces to which
have been immobilized ssDNA molecules.
They are used by hybridizing labeled ssDNA molecules to the DNA
on the Microarray, and assaying the for the labeled ssDNA molecules,
thereby determining the positions on the Microarray where hybridization
occurred.
Current DNA Microarray technology includes
two rather distinct methodologies:
1. immobilized single-stranded cDNA molecules: long, non-synthetic
substrates, at low density
2. immobilized short oligonucleotide ssDNA: short, synthetic substrates,
at high density
a. cDNA Microarrays:
ssDNA from a cDNA library is used and immobilized to a 2D solid surface, usually either a glass slide or a nylon membrane. Individual cDNA species are "spotted" in a 2D-grid on the solid surface. The number of spots is relatively small, at most about 80 x 80 or 6400 spots. These individual cDNA species might for example be obtained from separate Lambda Phage in a lambda cDNA library.
Experimentally, such arrays can be prepared at relatively low expense. They are then used to detect expression of cognate mRNAs (or via cDNAs from these cDNAs) by hybridizing the cDNAs obtained from some given experimental condition to the cDNAs immobilized on the surface. Since each of the 6400 spots represents expression of a different gene, one can assay expression of 6400 genes simultaneously. Further, using different fluors for lableled the experimental cDNA, e.g. lissamine-labeled vs fluorescein-labeled, one can assay in the same experiment two different experimental conditions.
Example:
cDNA microarray and stage-specific gene expression
in the malaria parasite Plasmodium falciparum
Figure 1: schematic of preparation of the cDNA array from PCR products (Fig 1b) and of the differential labeling of two-stages of malaria (trophozoite and gametocyte stages) mRNA with two fluoro-labels (one green, one red) prior to hybridization.
Figure 2: data examples and reproducibility
Second Example:
cDNA microarray of 10 Arabidopsis and 1046 Human cDNA species,
plus and minus Heat Shock treatment, color coded to shown heat
shock induction (red boxes) or repression (green boxes).
b. Oligonucleotide Microarrays:
To get really high densities of substrate ssDNA molecules on DNA microarrays, companies such as AffyMetrix build DNA microarrays using 'silicon valley' chip technology.
Oligonucleotides (short ssDNA molecules, e.g. 25 bp long ... or 25-mers) are synthesized on the chips. Synthesis technology is similar to that found in the organic chemistry technology associated with Oligonucleotide Synthesizers such as purchased from ABI / Perkin-Elmer via their GeneChips.
However: masks are used ... such that synthesis occurs only with light radiation ... and light can only pass through regions in the mask where there are spaces ... this technology permits synthesis of a high density of oligonucleotides (80,000 spots), each of a different sequence, on a chip much like a silicon chip.
cDNA species or mRNA species are then hybridized to such DNA chips ... or total DNA from an individual appropriately restriction digested, to determine SNP information about the individual ...
Usually, several oligos are used per gene, e.g. 5 oligos per gene. Thus, if the gene is expressed, mRNA, or corresponding cDNA, should hybridize to all 5 oligos. This provides controls: some oligos hybridize better then others ... one learns which oligos work best, for the next generation of oligo microarrays ... and at the same time some hybridization should occur to all 5 oligos...
This technology then permits automated analyses of the type for which DNA microarrays provide information.
For example, one can automate analysis of individual human beings for their heterozygosity as determine by their Single Nucleotide Polymorphisms.
Given that specific SNPs as associated to some degree with a given genetic disease, one can in automated fashion determine the propensity of any given individual for a given genetic disease. The pharmaceutical companies would have you believe that given such knowledge they can better design drugs and treatments for you, taking into consideration all of your genetic disease propensities. This includes when in your lifetime to start treating you for a disease you might get ...
That is: individual, cradle to grave, prescriptive life care ...
Example of Oligoarray to analysis SNPs (Single Nucleotide Polymorphisms):
For SNP microarrays, oligos corresponding to the two alleles are placed in two rows, one below the other. Note the five oligos, corresponding to five oligos for this gene.
| BIMM100 | Syllabus
| Sections / Off Hrs | Grading
Policy | DNASYSTEM
|
| Lectures | Journal
Articles | Study Qs | Lab
Techniques | Exams |
If you have problems or comments, send email to Doug Smith
