Transcript Lecture 2: Biology Review II

Lecture 2: Biology Review II
Date: 8/29/02
Overview/Review of:
Mapping
 Molecular techniques
 Markers

Genetic Mapping
 Definition: A genetic map is an ordering of
genes and markers in a linear arrangement
corresponding to their physical order along
the chromosome. Based on linkage.
 Definition: A physical map is an ordering of
landmarks on DNA, regardless of
inheritance. Measured in base pairs.
Marker
 Definition: A marker is a gene or piece of
DNA with easily identified phenotype such
that cells or individuals with different alleles
are distinguishable.
 e.g. a gene with known function
 e.g. a single nucleotide change in DNA
Polymorphism
 Definition: A polymorphism is a detectable
and heritable variation at a locus.
 Definition: A marker is polymorphic if the
most abundant allele comprises less than X%
of all alleles, usually 95%.
 Definition: A mapping population is a
population used to map genes.
Natural Populations
 Definition: Natural populations are those
where mating is not controlled by the
experimenter, though the experimenter can
choose who to observe.
 Only phenotype observable, genotype
sometimes unknown, phase is unknown.
 Knowns: allele frequencies, genotype
frequencies, amount of disequilibrium.
Hardy-Weinberg Equilibrium I
 Refers to the equilibrium achieved at a single
locus.
 Hardy-Weinberg Equilibrium (HWE) is
achieved when the allele frequencies and
genotype frequencies do not change from
generation to generation.
Hardy-Weinberg Equilibrium II
 Let pA and pB be the frequencies of allele A and B in
the population. Let pAA be the frequency of
genotype AA. Similarly, pAB and pBB are genotype
frequencies.
 Then HWE implies that
pAA = pA2
pAB = 2pApB
pBB = pB2
Measures of Polymorphism
l
H  1   p i2
P(heterozygote) =
i 1
Definition: Polymorphism Information
Content (PIC)
l
l
l
PIC  1   pi2  2  pi2 p 2j
i 1
i 1 j i 1
Uninformative Matings
AB X AB
½ are informative
1 AA : 2 AB : 1 BB
uninformative
informative
Classical Linkage Analysis
 A few markers.


Must have detectable variation.
Must be substantially variable in study
population.
 Controlled crosses: testcross, backcross,
double- haploid
 Well-defined parental lines.
Three-Point Testcross Design
dominant
recessive
testcross
X
X
F1
A A
b b
S S X t t
Y Y
z z
A
S
Y
F2
b
b b
t X t t
z
z z
b b
t S
z z
Three-Point Testcross Results
 Count the number of recombinant haplotypes
produced by F1 parent. Calculate the
recombinant fraction for each pair of genes.
1
2
3
1
--
0.19
0.03
2
--
--
0.15
3
--
--
--
Map for Three-Point Testcross
3
1
2
0.03
0.15
0.19
Backcross Design
new recombinant
self
self
F2
no more changes
Large-Scale Mapping
 Many genetic markers
 Steps of analysis:



pairwise linkage analysis
group into linkage groups
order markers in each linkage group
Comparative Mapping
 Compare maps of different species.
 Due to similarities, information can be
transferred between species.
 Information about how genomes evolve.
 Uses conserved loci rather than highly
variable loci.
Molecular Techniques: probes
5’ – …AAGCCTAGAGCCCTTAGCCAAAAG… – 3’
3’ – …TTCGGATCTCGGGAATCGGTTTTC… – 5’
denature
add probe 3’ – *ATCTCGGGAATC – 5’
hybridization
5’ – …AAGCCTAGAGCCCTTAGCCAAAAG… – 3’
*ATCTCGGGAATC
Molecular Techniques:
restriction enzymes
Definition: An endonuclease is an enzyme (protein that acts
as a catalyst to speed up the rate of a biochemical reaction) that
cleaves nucleic acid strands at internal sites (phosphodiester
bond).
Definition: A restriction endonuclease is an enzyme that cuts
DNA at specific sites that it recognizes.
EcoRI
5’ GAATTC 3’
3’ CTTAAG 5’
number of cut
sites = N/4b
Molecular Techniques: gel
electrophoresis
 DNA is negatively charged. Proteins can
also be charged.
 An electric current is passed through a
porous medium (agarose, acrylamide) and
molecules in the medium respond by moving
in electric field, but at different rates based
on size and charge.
Electrophoretic Gel
Molecular Technique: PCR I
5’
5’
Denaturation and hybridization
5’
5’
Elongation & denaturation
5’
5’
Molecular Technique: PCR II
Physical Maps
 Banding patterns on chromosomes
 In-situ hybridization



Denature metaphase chromosomes
Add radioactive or fluorescent probe
Visualize chromosomes
 DNA fragmentation
 DNA sequence: still not practical for all
organisms
DNA Fragmentation
• Larger fragments better (rare cutters; partial digestion)
• Find overlap by sequencing or hybridization.
DNA Vector I
 Definition: A cloning vector is a DNA
molecule that is capable of self-replicating.
Insert the fragment of foreign DNA to make
recombinant DNA.
DNA Vector II
 phage: virus that infects bacteria (5-25 kb).
 cosmid: Packaged in lambda phage and infects E.
coli (35-45 kb).
 yeast artificial chromosome (YAC): has telemere,
centromere, and replication origin (200-2000 kb).
 bacterial artificial chromosome (BAC)
 plasmid: extrachromosomal circular DNA
nonessential for cell survival.
How Many Clones?
Let N be the number of clones made.
Let NS be the number of nonoverlapping clones
needed to cover the full genome.
 NS 1

Pclone not in library   
 NS 
log 1  P 
N
log 1  1 
NS 

N
More: M. S. Waterman
Introduction to
Computational Biology:
Maps, Sequences, and
Genomes
Genetic Mapping Still Needed
 Even if the full sequence is known, mapping
is still necessary.
 There must be some way to correlate a
trait/phenotype with something on the
sequence.
Physical Mapping Still Needed
 Linkage maps lack resolution



Sample more people
Better statistics
Let recombination accumulate over many
generations.
 Even with most precise linkage map can
identify a gene to 1 cM (1 Mb in humans).
Morphological Markers
 Differences in shape, color, size, etc.
 Must have one-to-one correspondence with a
controlling gene.
Protein Markers
 Definition: An isozyme are proteins with
same enzymatic function but different
structural, chemical, or immunological
characteristics.
 Differences: amino acid composition, size,
modifications (e.g. phosphorylation).
 Differences visualized: gel electrophoresis,
mass spectrometry, etc.
DNA Marker: RFLP I
 Definition: RFLP is Restriction Fragment Length
Polymorphism.
 DNA digested with endonuclease.
 Separate fragments by electrophoresis.
 Denature strands.
 Transfer single-stranded DNA to durable membrane
and immobilize (Southern blot).
 Hybridize labeled probe to the blot.
 Visualize probe.
DNA Marker: RFLP II
 DNA polymorphisms that RFLP identifies:



mutation in the restriction site
mutation elsewhere to create restriction site
insertion/deletion of DNA
 RFLP markers are codominant
Mini- and Micro-Satellite
Markers
 Definition: minisatellites or VNTR (Variable
Number of Tandem Repeats) are tandem
repeates of sequences 9-100 bp long.
Detected by hybridization or PCR.
 Definition: microsatellites or SSR (Simple
Sequence Repeat) are direct tandem repeated
sequences of DNA of 1-6 bp.
STS and EST
 Definition: Sequence tagged sites (STS) is a
short unique fragment of DNA.
 Definition: Expressed sequence tags (EST)
are subsets of STSs from cDNA clones.
Represent transcribed genes (e.g. usually
proteins).
Single-Strand Conformational
Polymorphism (SSCP)
 Detects changes as small as 1 nucleotide in
more than 1000 bp.
 Single-stranded DNA is electrophoresed on
gel and migrates based on size and shape.
 Visualized by Southern blot with specific
fragment probe or PCR specific fragment and
visualize directly.
Random Amplified
Polymorphic DNA (RAPD)
 PCR with short probes that bind randomly to
sites in the genome.
 Good for genomes where little sequence
information is available.
 Band-present is dominant.
 Expected number of products = 2fN/16b
Amplified Fragment Length
Polymorphism (AFLP)
 Cut DNA with frequent- and rare-cutting
endonuclease
 Anneal adapters to the ends of the frequentcutter cut sites.
 Amplify off adapters with PCR. Use various
specific primers to amplify subsets of total.
 Visualize on denaturing polyacrylamide gel.
Choosing Markers
 High polymorphism.
 Clear interpretation.
 Quick typing and easy automation.
 Personal preference.