polymorphisms. monomorphism: section of dna where the nucleotide sequence is the same for everyone...
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Monomorphism: Section of DNA where thenucleotide sequence is the same for everyonein the population.
Polymorphism: Section of DNA with at leasttwo common nucleotide sequences (alleles)in the population.
“Common” is arbitrarily defined as a frequency of 1% or more.
Definitions
Mutant: Allele with a frequency of less than 1%.
Types of Polymorphisms:
I. Protein/enzyme polymorphisms (assay is for the gene
product, e.g., blood groups)
II. DNA polymorphisms (assay the DNA directly)
1. SNP: Single nucleotide polymorphisms2. Tandem repeat polymorphisms3. Structural polymorphisms (insertions, deletions,
inversions, etc.) CNV = Copy Number Variant4. Sequencing polymorphisms
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
Structural polymorphism: Insertion
..TTATGCCTAACTGACTTACCCT...
..TTATGCCTAACGTACCTGCTAGCTATACCTGACTTACCCT...
Deletion
Structural polymorphism: Deletion
..TTATGCCTAACGTACCTGCTAGCTATACCTGACTTACCCT...
Initial Sequence
..TTATGCCTAACCCATATCGATCGTCCATGTGACTTACCCT...
Inverted Sequence
Structural polymorphism: Inversion
..TTATGCCTAACGTACCTGCTAGCTAACGTACCAGCCCTG...
..TTATGCCTAACGTACCTGCTAG...
NOTE: Not all duplications have the exact nucleotidesequence. Two sections are said to be duplicates when90% of the sequence is identical.
Structural polymorphism: Duplication
(A) Section of a chromosome breaks off
CTGACTTACCCT.....AGTCGCTAGATCTA
..TTATGCCTAACGTACCTGCTAGCTATACCTGACTTACCCT...
CTGACTTACCCT...
..TTATGCCTAACGTACCTGCTAGCTATAC
(B) Broken segment attaches to another chromosome(often at a telomere)
Structural polymorphism: Translocation
Copy Number Variant (CNV)
Long (> 1 kb or > 10 kb) structural variant (insertion, deletion, duplication, inversion, etc).
NOTE: Defined by the technology used to detect them, not by any conceptual difference between themand a structural variant.
Sequencing polymorphisms arethe general case
SNPs
Tandemrepeats
Structuralpolys
Sequencingpolymorphisms
Tools in Molecular Genetics:1. Electrophoresis2. Probes3. Polymerase Chain Reaction4. DNA Arrays (Gene Chips)
GAATTC... GACTTC... GAATTC...
TTAAG... CCTTAAG...
Probe:Section of single-stranded DNA (or RNA) thatbinds to complementary DNA and carries a“lightbulb”
PCR: Polymerase Chain Reaction
Purpose =Make a lot of copies of a desiredpiece 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 CCG
TT T
TT
GG G
G
G
GATCCAG
GATCCAG
GATCCAG
GATCCAGC
CT
TA
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
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) are ordinary nucleotides. The second (colored letters) are special nucleotides (dideoxy 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.
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