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Types of Polymorphisms
I. Protein/enzyme polymorphisms
Blood groups
II. DNA Polymorphisms
1. Single Nucleotide Polymorphisms (SNP)
2. Tandem Repeat Polymorphisms
• Microsattelites, Short Sequence Repeats (SSR)
• Variable number of tandem repeats (VNTR)
3. Structural Variants
• Insertion/Deletion/Inversion/Duplication/Translocation
• Copy Number Variants (CNV)
SNP: Single Nucleotide Polymorphism
Section of DNA that difference in one and
only one nucleotide.
Allele 1: C
TGATCTTG...........TGCCAGTT . . . . . . . . . CCGTAGCGAA
TGATCTTG...........TGCTAGTT . . . . . . . . . CCGTAGCGAA
Allele 2: T
Tandem Repeat Polymorphisms:
A nucleotide sequence is repeated over and
over again and the polymorphism is in the
number of times it is repeated.
Repeated sequence = GAAC
tandem repeat (8 repeat allele)
..TTATGAACGAACGAACGAACGAACGAACGAACGAACTTACGT...
..TTATGAACGAACGAACGAACTTACGT...
tandem repeat (4 repeat allele)
Insertion Polymorphism
..TTATGCCTAACTGACTTACCCT...
..TTATGCCTAACGTACCTGCTAGCTATACCTGACTTACCCT...
Insertion
Deletion Polymorphism
Deletion
..TTATGCCTAACGTACCTGCTAGCTATACCTGACTTACCCT...
..TTATGCCTAACTGACTTACCCT...
Inversion Polymorphism
Initial Sequence
..TTATGCCTAACGTACCTGCTAGCTATACCTGACTTACCCT...
..TTATGCCTAACCCATATCGATCGTCCATGTGACTTACCCT...
Inverted Sequence
Duplication Polymorphism
..TTATGCCTAACGTACCTGCTAG...
..TTATGCCTAACGTACCTGCTAGCTAACGTACCAGCCCTG...
NOTE: Not all duplications have the exact nucleotide
sequence. Two sections are said to be duplicates when
90% of the sequence is identical.
Translocation Polymorphism
(A) Section of a chromosome breaks off
..TTATGCCTAACGTACCTGCTAGCTATACCTGACTTACCCT...
..TTATGCCTAACGTACCTGCTAGCTATAC
CTGACTTACCCT...
(B) Broken segment attaches to another chromosome
(often at a telomere)
..AGTCGCTAGATCTA CTGACTTACCCT...
Copy Number Variant (CNV)
(1) Somewhat long (> 1kb) section of DNA that
is repeated throughout the genome with
variable copy numbers.
(2) Repeats do not have to be in tandem.
(3) Includes long insertions, deletions, and
duplications.
Redon et al. (2006). Nature 444(23), 444-454.
Tools in Molecular Genetics:
1. Electrophoresis
2. Probes
3. Polymerase Chain Reaction
4. Restriction Enzyme
5. Dideoxy Nucleotides
6. DNA Arrays (Gene Chips)
Electrophoresis:
start lanes
-
current
+
gel
timer
http://www.ucl.ac.uk/~ucbhjow/b241/biochemical.html
Probe:
Section of single-stranded DNA (or RNA) that
binds to complementary DNA and carries a
“lightbulb”
TTAAG...
GAATTC...
CTTAAG...
GACTTC...
C
GAATTC...
PCR: Polymerase Chain Reaction
Purpose =
Make a lot of copies of a desired
piece of DNA (i.e., “amplify” the DNA)
PCR: Polymerase Chain Reaction
(1)
(2)
(3)
(4)
Start with a soup containing:
the DNA that you want to amplify
enzymes to replicate DNA (polymerase)
lottsa free nucleotides
primers = short initial section of the gene that you want
to amplify (e.g., GATCCAG )
G G
A
A
G
T A
C
T
GATCCAG
GATCCAG C T
A
C
T GATCCAG
C
T
G
GATCCAG
G
C
G
T C
A
T
C
G
PCR: Polymerase Chain Reaction
Procedure:
1. Heat the mixture. Just before the boiling point of
water, the DNA will become single-stranded.
2. Cool the mixture. As the mixture cools, the primer will
bind to the DNA and the polymerase will synthesize a
new strand for each strand of DNA.
3. Repeat steps 1 and 2 until a sufficient amount of the
desired gene is available for analysis
(a)
(b)
Primers
(c)
New
Strands
Free
Nucleotides
PCR: Polymerase Chain Reaction
http://www.britannica.com/nobel/cap/opolchr001a4.html
RFLP:
Restriction Fragment Length Polymorphism
Polymorphism based on whether or not a restriction
enzyme cuts a section of DNA. Usually two alleles: (1)
the restriction enzyme does cut in the middle of the
genes; and (2) the restriction enzyme does not cut in
the middle of the gene.
restriction enzyme = enzyme that recognizes a specific
nucleotide sequence and cuts the DNA at that sequence.
Allele 1:
GAATTC...
GACTTC...
GAATTC...
restriction enzymes
AATTC...
GACTTC...
G
GAATTC...
GAATTC...
GAATTC...
Allele 2:
AATTC...
G
AATTC...
G
(c)
Probe:
TTAAG...
AATTC...
CTTAAG...
GACTTC...
C
G
CTTAAG...
AATTC...
C
G
CTTAAG...
G
C
Probe:
TTAAG...
Probe:
TTAAG...
AATTC...
Starting Lanes
Moe
Homozygote:
neither allele cut, so
two long
strands
Larry
Curly
Heterozygote:
One long strand from the
allele that was not cut, and
two strands from the allele
that was cut
Homozygote:
two alleles which
the restriction
enzyme cut
The “Olden” Days
Current Technology
1. PCR that includes dideoxy nucleotides
with ordinary nucleotides.
2. Have a laser scan the fragments from
electrophoresis.
dideoxy nucleotides = “color coded” nucleotides (e.g., A, T, C,
G) that stop the synthesis of a new DNA chain when they are
inserted into the chain.
Finding a SNP
http://www.sanger.ac.uk/genetics/exon/
SNP Genotyping Machine
(7000 genotypes per day)
http://www.wtcrf.ed.ac.uk/genetics/images/snp1.jpg
Wellcome Trust Clinical Research Facilities
Potential New Method:
Nanopores
How the
Human Genome
was Sequenced:
(See Text)
1. Heat the DNA to make it single stranded and add a primer. The primer binds to its
complementary sequence in the DNA.
DNA strand to sequence
ATGACCTCG??????????????
TACTGGAGC
Primer
?
2. Add nucleotide alphabet soup. Two types of nucleotides are in the soup. The first (black letters) are
ordinary nucleotides. The second (colored letters) are special nucleotides that have two important
properties: (1) they will halt the synthesis of the DNA strand whenever they are incorporated into
it, and (2) they will fluoresce when viewed under the appropriate lighting.
T
C
A
G
A
C
C
T
A
C G
G
A
T
G
T
T
DNA strand to sequence
C
A
C
G
A
A
A
T
C
ATGACCTCG??????????????
?
T
TACTGGAGC
C
G
A
C
A
C
T
A
Primer
G
T
A
T
G
A
A
G
C
T
G
T
C
G
C
T
G
T
A
C
G
T
C
G
T
A
3. Add the polymerase (an enzyme that adds free nucleotides to the primer strand). The polymerase
will “grab” free nucleotides and add the appropriate one to the extend the strand.
T
C
A
G
A
C
C
T
A
C G
A
T
G
T
T
DNA strand to sequence
G
C
A
C
G
A
A
Polymerase
A
T
C
ATGACCTCG??????????????
?
T
TACTGGAGC AA C
G
A
C
C
T
A
Primer
G
T
A
T
A
G
A
A
G
C
T
G
T
C
G
C
T
T
G
A
C
G
T
C
G
T
A
4. Complementary strands will be synthesized, but they will be of different lengths depending
on where the colored nucleotide is incorporated. Eight examples are given below.
ATGACCTCGTAGGCAAAGCCTCG
TACTGGAGCA
T
ATGACCTCGTAGGCAAAGCCTCG
TACTGGAGCATCCG
T
ATGACCTCGTAGGCAAAGCCTCG
TACTGGAGCAT
T
ATGACCTCGTAGGCAAAGCCTCG
TACTGGAGCATCCGTT
T
ATGACCTCGTAGGCAAAGCCTCG
TACTGGAGCATC
T
ATGACCTCGTAGGCAAAGCCTCG
TACTGGAGCATCCGTTTCG
T
? ATGACCTCGTAGGCAAAGCCTCG
TACTGGAGCATCCGTTTCGGAAA
T
A
ATGACCTCGTAGGCAAAGCCTCG
TACTGGAGCATCC
5.Heat the DNA to make it single-stranded. There will be many copies of the template strand and
also many copies of different length of the synthesized strands.
TACTGGAGCATCCGTTTCG
ATGACCTCGTAGGCAAAGCCTCG
TACTGGAGCA
?
TACTGGAGCATCCGTTTC
TACTGGAGCATCC
TACTGGAGCATCCGTTTCGGAAA
TACTGGAGCATCCGT
TACTGGAGCAT
ATGACCTCGTAGGCAAAGCCTCG
TACTGGAGCATC
TACTGGAGCATCCGTT
TACTGGAGCATCCGTTTCGGA
TACTGGAGCATCCGTTTCGGAA
A
ATGACCTCGTAGGCAAAGCCTCG
TACTGGAGCATCCG
?
TACTGGAGCATCCGTTTCG
?
6. Use electrophoresis to separate the strands according to size.
TACTGGAGCATCCGTTTCGGAAA
TACTGGAGCATCCGTTTCGG
TACTGGAGCATCCGTTTCG
TACTGGAGCATCCGTTTC
TACTGGAGCATCCGTTT
TACTGGAGCATCCGTT
TACTGGAGCATCCGT
TACTGGAGCATCCG
TACTGGAGCATCC
TACTGGAGCATC
TACTGGAGCAT
TACTGGAGCA
A
7. Viewing the gel under a special light allows the colored nucleotides to fluoresce. This
lights up the band. The color-coding permits the DNA sequence to be read.
TACTGGAGCATCCGTTTCGGAAA
TACTGGAGCATCCGTTTCGG
TACTGGAGCATCCGTTTCG
TACTGGAGCATCCGTTTC
TACTGGAGCATCCGTTT
TACTGGAGCATCCGTT
TACTGGAGCATCCGT
TACTGGAGCATCCG
TACTGGAGCATCC
TACTGGAGCATC
TACTGGAGCAT
TACTGGAGCA
A
(a)
AATGCGTCATG
ATGCCTGAAATGC
CGTTACGTGATGATGCC
(b)
AATGCGTCATG
CGTTACGTGATGATGCC
ATGCCTGAAATGC