| 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. Genetic Maps vs Physical Maps
Comparison of Genetic and Physical Maps: ... [Brown, Fig. 3.1]
Genetic Map is colinear with physical placement of Genes on DNA but quantitation is difficult due to variations of Recombination processes between organisms (more rare in eukaryotes than in prokaryotes) and due to dependence on DNA sequence (hot spots for recombination)
Physical Map of Genome: placement of Genes and Sites on Nucleotide Sequence of DNA, i.e. on the physically real Genetic Material
The Ultimate Physical Map is the Nucleotide Sequence ...
Note to serve as a Marker in either a Genetic Map (genes, map via Recombination Frequency) or a Physical Map (Sites on DNA, map via locating the site on the DNA), a Gene or Site must occur in more than one state. Such states are called Alleles.
In classical genetics, the results or manifestations of one state or another, i.e. one Allele or another, of a given Gene are called the Phenotype associated with a given Allele. Such might be eye color or pea shape. The differences in the Gene itself associated with a given Allele are called the Genotype.
In modern genetics, the Genotype
is a change in the DNA sequence encoding the given Gene.
The classical Phenotype such as the examples just given arise
from changes in the amino acid sequence of the Protein expressed
by the given Gene, resulting in change in activity of the Protein
(perhaps no activity for one Allele of the Gene), resulting in
change in a metabolic pathway, resulting in change in eye color
or pea shape.
In modern genetics, the Phenotype can also be defined at the DNA
level. The Phenotype is any measure of the results of one Allele
or another. Thus, the Phenotype can also be the change in the
DNA sequence associated with the change from one Allele of a Gene
to another, because with appropriate sequencing one can determine
this change in DNA sequence.
This ability to define the
classical genetic concept of Genotype in terms of specific
molecular changes in the genetic material or DNA molecules, coupled
with ability to measure these specific molecular changes via DNA
sequencing, bring the concepts of Genotype and Phenotype
into close proximity to each other.
B. DNA Markers for Genetic Mapping - Polymorphisms
Genetic Marker: mutant vs "wild
type" ... compare 2 phenotypes ... Alleles
Based on Mutation eliminating (or creating) a R.site
Polymorphisms: individuals in a populations have different
Alleles or variants
of a given Gene Marker
What is an Allele? ... some change in a Gene or Site that can be assayed: phenotype
In principle, any DNA base
change is now an Allele
... can "assay" any such change via DNA Sequencing ...
Uses:
1. Markers for Genetic Mapping: Pedigree Analysis
2. Markers for Physical Mapping: Restriction Mapping
3. Genetic disease: compare Diseased vs Non-Diseased individuals:
4. Forensic purposes: compare DNA from blood from victim
and suspect,
compare with blood at "scene of the crime"
5. Identification purposes: compare DNA from blood at some
site with DNA stock
=> military, accidents, etc
6. Parentage identification ... "who's the father?"
... Dogs, Horses, other animals
1. Restriction Fragment
Length Polymorphisms (RFLPs)
... [Brown, Fig. 2.4]
R.sites as DNA markers can serve as Genome sites for Genetic Mapping
Change in a R.site: assay via cleavage - or not - by R.enzyme ... bands on gels
A change in a Restriction Site:
one allele - the R.Site can be cleaved
another allele - the R. Site can NOT be cleaved
These two alleles yield two different sizes for R.fragments
... i.e. changes in the Restriction Fragment Length
Thus: Restriction Fragment Length Polymorphisms
NOTE that no Phenotype is associated here with the biology of
the organism!!
The "phenotype" is defined by Assays in the Lab ...
Additional Examples of RFLPs can be found here, including a Pedigree Analysis example.
2. Simple Sequence Length Polymorphisms (SSLPs)
These arise from Repeated Sequences present in DNA genomes of higher organisms
Repeated Sequences are of two general types:
1. MiniSatellites or
VNTRs (Variable Number of Tandem Repeats)
These are regions of DNA containing tandem repeats
where the repeats are in the size range 25 bp to a few hundred
base pairs in length.
2. MicroSatellite or
Simple Tandem Repeats (STRs)
These are also tandem repeat regions of DNA, but where the
repeat sizes are smaller, generally 2 to 7 bp in length
Polymorphisms arise in populations of the organism
either by variations in
1. the precise sequence found in each repeat
2. in the number of repeats found in a region of the repeat
The latter type of polymorphism is currently used more often than the former. Each such naturally occuring polymorphism in a population of the organism is an Allele for the site on the DNA at which the repeat region occurs. Such a polymorphism arising from variations in the number of repeats results in a change in the length of the repeated region, and is called an SSLP.
Measurement of SSLPs - Allelic
Phenotypes: ...[Brown, Fig 2.5]
... determine the length of the SSLP in a given DNA sample via
its size using Gel Electrophoresis
The MicroSatellite map of the Human Genome is shown in Brown, Research Brief 2.1
3. Single Nucleotide Polymorphisms (SNPs) ... [Brown, Fig 2.6]
These are Polymorphisms or Alleles occurring naturally in a population resulting from point mutations or changes in single nucleotides in the DNA in individuals in the population. They usually are changes within a Gene, resulting in a change in an amino acid of the encoded Protein, resulting in a change in a pathway, yielding a Phenotype such as a genetic disease in humans.
Note that SNPs that occur in a R.site, resulting in Alleles that can be cut or not by the R.enzyme, also result in RFLPs.
Discovery of SNPs:
Unless a SNP happens to fall within a R.site,
and therefore also results in an RFLP, the only way to discover
naturally-occuring SNPs is by way of DNA sequencing.
Considerable effort is currently underway in many laboratories
and biotech companies to discover SNPs associated with human genetic
disease.
It is instructive to have a look at the SNP database at NCBI.
C. Methodology - Polymerase Chain Reaction (PCR)
1. DNA Polymerases - Paradigm:
DNA Polymerase I of E. coli ... PolI
... [Brown, p. 311-313]
Pol I was the first DNA polymerase
enzyme to be isolated and characterized.
Single polypeptide chain, 109 kD, 3 domains corresponding to principle
activities
Has the following interesting
properties:
1. Several enzymatic activities ... the 3 major activities:
a. Polymerization: 5' to 3' only C-terminal domain; "claw"
3D structure
b. Exonuclease: 3' to 5' ... Proof-Reading Middle domain
c. Exonuclease: 5' to 3' ... Nick Translation N-terminal
domain
Klenow fragment: lacks the N-term 5'-3' Exo domain
2. Polymerization Reaction:
a. DNA substrate must have
3 properties:
1. Template strand
2. Primer strand
3. Primer Terminus: 3'-OH
b. Reaction: DNA is used to make more DNA
Precursor: dNTPs, N = G,A,T,C
dNMPs incorporated into growing DNA strand; PPi (pyrophosphate)
released
3. Proof-Reading: 3' -> 5' Exonuclease Reaction:
When PolI makes an error in polymerization, the 3'->5' Exonuclease
removes the incorrect dNMP, permitting PolI to try again ...
Decreases the Error of Incorporation rate from 10-5 to around
10-7
4. Nick Translation: 5' -> 3' Exonuclease Reaction:
Combined 5'->3' Polymerization with 5'->3' Exonuclease ...
"translation" of nick
Used physiologically in DNA Repair and during DNA replication
Elongation
Highly useful in the lab to make radioactively-labeled DNA:
Introduce nicks with Pancreatic DNase
Nick translate with PolI using Alpha-P32 - labeled
dNTP precursor
2. Polymerase Chain Reaction
(PCR) ...[Brown,
Tech Notes 2.2]
Combination of Polymerization with Denaturation-Renaturation Hybridization
PCR procedure:
1. Have Two Primers to a DNA
fragment, one to each Strand,
which hybridize 500-2000 bp apart from each other.
2. Concentration of Primers is very high compared with DNA ... Primers in Excess
3. Denature the DNA (95·C, 1 min)
4. Hybridize the Primers (eg 67·C, 2 min)
5. Extend the Primers via DNA
synthesis using a heat-resistant DNA polymerase
eg Taq polymerase (eg 75·C, 3 min)
6. Now have doubled the DNA present ...
7. Repeat steps 2-5 for 20-30 times ("cycles") via a heat-to-95·C : cool-to-67·C procedure
Each cycle doubles the amount
of DNA present !!
Thus, the number of DNA molecules increases EXPONENTIALLY with
numbers of cycles of PCR.
Nearly all of these molecules (> 99% after 12 cycles) are Target DNA molecules, i.e. contains only primer nucleotides and nucleotides found between the primers.
After 30 cycles, the amplification
in total DNA molecules is 230.
Since 210 ~ 1000, 230 ~ 1000 x 1000 x 1000
= 1,000,000,000 = 109
There is thus an approximate billion-fold amplication in
total DNA molecules.
All but 60 of these are Target DNA molecules.
Detailed calculations of Target DNA molecules synthesized as a function of number of cycles of PCR, together with Figures, can be found here.
Another aid to understanding
PCR can be found here.
Can use PCR for many purposes, eg DNA sequencing:
1. Use ONLY one Primer, with enough initial DNA for sequencing
2. Extend with DNA polymerase in presence of ddNTPs
Only one strand in made, and made as a Nested Set of Chain
Terminated fragments ...
PCR can also be used for amplification of mRNA, etc etc ...
PCR is often used to define
STSs (Sequence Tagged Sites).
That is, in defining the STS, the sequences of the two PCR primers
are given, and the size of the PCR product. Thus, any investigator
can make their own STS probe by purchasing the two primers and
then doing their own PCR reaction with appropriate genomic DNA.
An example of an STS for human chromosome 18, defined in this manner as a PCR product, can be found here.
D. Pedigree Analysis - Heterozygosity
Some Lab Technique assistance on human Genetic Mapping can be found here.
A large amount of information is available at NCBI on Human Genome resources, and in particular on Human Genome maps
Human Genetics:
Human genetics: limited by inability to do
genetic crosses with human beings.
Need to work with data that comes from analysis of naturally-occuring
Human Pedigrees:
genetic data of children, parents, grandparents over generations.
Traditional data: limited to genetic disease and human phenotypic
data
More recently: advent of Physical Markers
for Human Genetics
Physical Markers: DNA sites - RFLPs, Microsatellites,
SNPs, ESTs, Sequence data per se
Use of Physical Markers has revolutionized
Human Genetics
Genome DNA sequence data is bringing in line the human Genetic
Maps with real Physical Maps of the Human Chromosomes ... compare
Brown, Research Brief 2.1 with Research Brief 3.1
Examples of Pedigrees:
1. Some problems with Pedigree analysis are shown in Brown, Fig 2.14
2. An example of a Pedigree for an RFLP locus is shown here.
This latter example also permits calculation of the Heterozygosity for the RFLP DNA marker shown and for the data from the 14 individuals in the Pedigree shown.
Heterozygosity: fraction of individuals heterozygous for the given DNA marker.
To be useful in genetic analysis, markers must have a reasonably high Heterozygosity. Markers with low heterozygosity are of no value in Linkage analysis.
For example, consider a marker with 0 heterozygosity (all individuals are homozygous for the marker); such a marker gives no useful data if one attempts to genetically map this marker relative to another marker.
| 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