Types of Polymorphisms
I. Protein/enzyme polymorphisms
Blood groups
II. DNA Polymorphisms1. Single Nucleotide Polymorphisms (SNP)2. Tandem Repeat Polymorphisms
3. Structural Variants• Insertion/Deletion/Inversion/Duplication/Translocation• Copy Number Variants (CNV)
• Microsattelites, Short Sequence Repeats (SSR)• Variable number of tandem repeats (VNTR)
SNP: Single Nucleotide Polymorphism
Section of DNA that difference in one and only one nucleotide.
TGATCTTG...........TGCCAGTT . . . . . . . . . CCGTAGCGAA
TGATCTTG...........TGCTAGTT . . . . . . . . . CCGTAGCGAA
Allele 1: C
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.
..TTATGAACGAACGAACGAACGAACGAACGAACGAACTTACGT...
..TTATGAACGAACGAACGAACTTACGT...
tandem repeat (8 repeat allele)
tandem repeat (4 repeat allele)
Repeated sequence = GAAC
..TTATGCCTAACTGACTTACCCT...
..TTATGCCTAACGTACCTGCTAGCTATACCTGACTTACCCT...
Insertion
Insertion Polymorphism
..TTATGCCTAACTGACTTACCCT...
..TTATGCCTAACGTACCTGCTAGCTATACCTGACTTACCCT...
Deletion
Deletion Polymorphism
Inversion Polymorphism
..TTATGCCTAACGTACCTGCTAGCTATACCTGACTTACCCT...
Initial Sequence
..TTATGCCTAACCCATATCGATCGTCCATGTGACTTACCCT...
Inverted Sequence
Duplication Polymorphism
..TTATGCCTAACGTACCTGCTAGCTAACGTACCAGCCCTG...
..TTATGCCTAACGTACCTGCTAG...
NOTE: Not all duplications have the exact nucleotidesequence. Two sections are said to be duplicates when90% of the sequence is identical.
Translocation Polymorphism
(A) Section of a chromosome breaks off
CTGACTTACCCT.....AGTCGCTAGATCTA
..TTATGCCTAACGTACCTGCTAGCTATACCTGACTTACCCT...
CTGACTTACCCT...
..TTATGCCTAACGTACCTGCTAGCTATAC
(B) Broken segment attaches to another chromosome(often at a telomere)
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.
Tools in Molecular Genetics:1. Electrophoresis2. Probes3. Polymerase Chain Reaction4. Restriction Enzyme5. Dideoxy Nucleotides6. DNA Arrays (Gene Chips)
GAATTC... GACTTC... GAATTC...
TTAAG... CCTTAAG...
Probe:Section of single-stranded DNA (or RNA) that
binds to complementary DNA and carries a“lightbulb”
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
Start with a soup containing:(1) the DNA that you want to amplify(2) enzymes to replicate DNA (polymerase)(3) lottsa free nucleotides(4) primers = short initial section of the gene that you want
to amplify (e.g., )
CA
AA
C C CC GTT T
T T
GG G
G
G
GATCCAG
GATCCAG
GATCCAG
GATCCAG C
CT
T A
A
GATCCAG
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
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.
GAATTC... GACTTC... GAATTC...
AATTC... GACTTC... G
Allele 1:
restriction enzymes
GAATTC... GAATTC... GAATTC...Allele 2:
AATTC... G
AATTC... G
AATTC... GACTTC... G TTAAG...
Probe:
CCTTAAG...
AATTC... G
TTAAG...Probe:
CCTTAAG...
AATTC... G
TTAAG...Probe:
CCTTAAG...
(c)
Starting Lanes
Moe Larry Curly
Homozygote:neither allele cut, so
two longstrands
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
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.
http://www.wtcrf.ed.ac.uk/genetics/images/snp1.jpg
SNP Genotyping Machine(7000 genotypes per day)
Wellcome Trust Clinical Research Facilities
TACTGGAGC
ATGACCTCG?????????????? ?
DNA strand to sequence
Primer
1. Heat the DNA to make it single stranded and add a primer. The primer binds to its complementary sequence in the DNA.
2. Add nucleotide alphabet soup. Two types of nucleotides are in the soup. The first (black letters) areordinary nucleotides. The second (colored letters) are special nucleotides that have two importantproperties: (1) they will halt the synthesis of the DNA strand whenever they are incorporated intoit, and (2) they will fluoresce when viewed under the appropriate lighting.
TACTGGAGC
ATGACCTCG?????????????? ?
DNA strand to sequence
PrimerAAA
A
A
A
A
AA
A
A
A
A
A
A
A
TT
TT
T
T
T
TT
T
T
T
T
T
T
T
CC
C
C
CC
C
C
C
C
C C C
C
CG
G
G
G
G
G
G G
G
G
G
G
G
G
3. Add the polymerase (an enzyme that adds free nucleotides to the primer strand). The polymerasewill “grab” free nucleotides and add the appropriate one to the extend the strand.
TACTGGAGC
ATGACCTCG?????????????? ?
DNA strand to sequence
PrimerAAA
A
A
A
A
AA
A
A
A
AA
A
A
TT
TT
T
T
T
TT
T
T
T
T
T
T
T
CC
C
C
CC
C
C
C
C
C C C
C
CG
G
G
G
G
G
G G
G
G
G
G
G
G
AA
Polymerase
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.
TA
ATACTGGAGC
ATGACCTCG GGCAAAGCCTCG T
TA
ATACTGGAGC
ATGACCTCG GGCAAAGCCTCG T
T
TA
ATACTGGAGC
ATGACCTCG GGCAAAGCCTCG T
TC
TA
ATACTGGAGC
ATGACCTCG GGCAAAGCCTCG ?
TCC
TA
ATACTGGAGC
ATGACCTCG GGCAAAGCCTCG T
TCCG
TA
ATACTGGAGC
ATGACCTCG GGCAAAGCCTCG T
TCCGTT
TA
ATACTGGAGC
ATGACCTCG GGCAAAGCCTCG T
TCC TTTCG G
TA
ATACTGGAGC
ATGACCTCG GGCAAAGCCTCG T
TCC TTTCG GGAAA A
5.Heat the DNA to make it single-stranded. There will be many copies of the template strand andalso many copies of different length of the synthesized strands.
ATACTGGAGC
?TAATGACCTCG GGCAAAGCCTCG
T AACTGGAGC T T AACTGGAGC TC
ATACTGGAGC TCC
ATACTGGAGC TCCG
ATACTGGAGC TCCGT
ATACTGGAGC TCC TTTCG G
ATACTGGAGC TCC TTTCG GGA
ATACTGGAGC TCC TTTCG GGAAA A
?TAATGACCTCG GGCAAAGCCTCG
ATACTGGAGC TCC TTTCG GGAA
ATACTGGAGC TCC TTTCG
ATACTGGAGC TCC TTG
ATACTGGAGC TCC TTTCG G
?TAATGACCTCG GGCAAAGCCTCG
6. Use electrophoresis to separate the strands according to size.
ATACTGGAGC
T AACTGGAGC T
T AACTGGAGC TC
ATACTGGAGC TCC
ATACTGGAGC TCCG
ATACTGGAGC TCCGT
ATACTGGAGC TCC TTTCG G
ATACTGGAGC TCC TTTCG GG
ATACTGGAGC TCC TTTCG GGAAA A
G CATACTGGA C TC TTTG
ATACTGGAGC TCC TTG
ATACTGGAGC TCC TTTCG
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.
ATACTGGAGC
T AACTGGAGC T
T AACTGGAGC TC
ATACTGGAGC TCC
ATACTGGAGC TCCG
ATACTGGAGC TCCGT
ATACTGGAGC TCC TTTCG G
ATACTGGAGC TCC TTTCG GG
ATACTGGAGC TCC TTTCG GGAAA A
G CATACTGGA C TC TTTG
ATACTGGAGC TCC TTG
ATACTGGAGC TCC TTTCG