nucleic acid variation DNA POLYMORPHISMS

• indirect • AMPLIFICATION INDIRECT ESTIMATES

– DNA/DNA hybridization OF SEQUENCE DIVERGENCE
– enzymatic degradation – PCR BASED MEASURES
• restriction enzyme digestion (RFLP) • RAPD
– amplification polymorphism (RAPD) – VARIATION IN PRIMER SITES

– repeat sequence variation (VNTR / STR) • STS

– SEQUENCE TAGGED SITES with RFLP

• direct
– sequencing

RAPD
randomly amplified polymorphic DNA
• PCR based technique
– use of single short oligonucleotide primer -
often 10 base
PRIMER 1

PRIMER 1

RAPD RAPD
randomly amplified polymorphic DNA randomly amplified polymorphic DNA
• PCR based technique • Multiple “loci” amplify
– use of single short oligonucleotide primer - – many different locations of 10 bp primer
often 10 base site
PRIMER 1 • Any location with inverted repeat can
amplify
PRIMER 1

primers located as inverted repeats PRIMER 1

PRIMER 1
 RAPD RAPD
randomly amplified polymorphic DNA randomly amplified polymorphic DNA
• Multiple “loci” amplify • “+” alleles show dominance
– nucleotide changes in one or both sites can +/+
“destroy” a band and produce a
polymorphism

PRIMER 1 no amplification !

RAPD RAPD
randomly amplified polymorphic DNA randomly amplified polymorphic DNA
• “+” alleles show dominance • “+” alleles show dominance
• “-” allele is recessive +/+ +/-
• “-” allele is recessive +/+ +/- -/-

X
- / - HOMOZYGOTES PRODUCE
X NO PRODUCT
X

RAPD
patterns

segregating segregating
band bands
RAPD patterns from RAPD patterns
Capsella bursa-pastoris from common
carp
Cyprinus carpio

RAPD
polymorphisms

Y-W Yang, ET AL. Bot. Bull. Acad. Sin. (1998) 39: 17_21

RAPD RAPD
randomly amplified polymorphic DNA randomly amplified polymorphic DNA
• “+” alleles show dominance • Can be used to screen a genome quickly
• “-” allele is recessive • Many highly variable bands often seen
– dominance limits ability to calculate allele
frequency
– conditions must be carefully controlled
• RAPD can produce spurious amplification
– number of nucleotide differences between
• Frequencies estimated from frequency of “alleles” unknown
recessives (-), assuming HWE

DNA POLYMORPHISMS HOW DO WE VISUALIZE RFLP?

• AMPLIFICATION INDIRECT ESTIMATES • Use sequence with large amounts of

OF SEQUENCE DIVERGENCE DNA
– PCR BASED MEASURES – mtDNA, cpDNA or rRNA
• RAPD • (limited # sequences)
– VARIATION IN PRIMER SITES
• STS • use Southern blotting with cloned probes
– SEQUENCE TAGGED SITES with RFLP • use PCR
 STS
• SEQUENCE TAGGED SITE
– site with unique location in the genome
which can be identified by amplification
using a specific pair of PCR primers
• Primers developed to be STS locus specific

STS Combine STS with RFLP

COMBINATION OF PRIMER 1 + PRIMER 2
REPRESENTS UNIQUE SITE

PRIMER 1 PRIMER 1

PRIMER 2 polymorphism PRIMER 2

Restriction recognition sites

Combine STS with RFLP STS-RFLP PROBLEM:

RFLP + RFLP - • STS products are usually small (<500 bp)
• Limited degree of nucleotide polymorphism
Patterns for – ( < 1/100? nucleotides variable)
previous PCR
fragments
following
restriction
 nucleic acid variation DNA VARIATION
• indirect • RESTRICTION SITE VARIATION
– DNA/DNA hybridization – “RANDOM SITES” - RFLP
– enzymatic degradation – TARGETED SEQUENCES
• restriction enzyme digestion (RFLP) • PCR - STS
– amplification polymorphism (RAPD) – SPECIAL SIZE VARIATION
– repeat sequence variation (VNTR / STR) • VNTR
• direct • STR
– sequencing

VNTR LOCI
• Variable Number of Tandem Repeats
– hypervariable loci or minisatellite sequences
• Special kind of RFLP sequence
• variation in number of repeats of short
(30-300 bp) core segment between
constant restriction sites.
 VNTR LOCI VNTR LOCI
VNTR LOCI
• "mutational" process probably involves • VNTR probes:
unequal cross-over or replication slippage – Multilocus probe: Jeffreys' probes
– core sequence hybridizes to several (many)
• High levels of polymorphism different locations
– MANY alleles – not utilized widely in human genetics
• gene frequency estimation difficult

• usually visualized by Southern blotting

DNA Multilocus VNTR Patterns

VNTR family DNA Forensics: Paternity
study
from birds Shown are DNA "fingerprints" from
a new mother (M), her recently born
child (C), and two possible fathers
(F1, and F2). The mother and the
child share some DNA bands since
they also share 50% of their genes
but they each have bands that are
not represented among the bands of
the other.

The two possible fathers have

dissimilar DNA banding patterns.
The question is does one of the
possible fathers have several bands
similar to the child which are not
shared with either the mother or the
other possible father.
Dowling et al, 1996

DNA Multilocus VNTR Patterns

from four sets of twins
Shown are DNA banding VNTR LOCI
patterns from four sets of
twins. If the band patterns
are identical, then the twins • VNTR probes:
should be identical – Single Locus probes
(maternal) but if the band
patterns of two twins are – core sequence hybridizes to one unique
different, then the twins are locus in genome
non-identical (fraternal).
Non-identical twins should
– preferred VNTR sequences because variation
share some of their DNA can be assigned to locus
bands since they share 50%
of the genes.
 single locus vntr patterns

Serial Rape Case - 3 suspects, evidence from 7 victims

VNTR limitations VNTR applications

• may be difficult to score size of "allele" • DNA fingerprinting - forensic

accurately
• limited number of VNTR loci
• non-random distribution in genome
• paternity analysis
• analysis of linkage to other loci
– (limited because of nonrandom locus
distribution in genome)