Polymorphisms. Monomorphism: Section of DNA where the nucleotide sequence is the same for everyone in the population. Polymorphism: Section of DNA with.

Polymorphisms

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...


Deletion

Structural polymorphism: Deletion


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


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.

Redon et al. (2006). Nature 444(23), 444-454.

Sequencing polymorphisms arethe general case

SNPs

Tandemrepeats

Structuralpolys

Sequencingpolymorphisms

Tools in Molecular Genetics:1. Electrophoresis2. Probes3. Polymerase Chain Reaction4. DNA Arrays (Gene Chips)

+gel timer

current

start lanes-

Electrophoresis:

http://www.ucl.ac.uk/~ucbhjow/b241/biochemical.html

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)


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


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)Primers(b)

NewStrands

FreeNucleotides

(c)

http://www.britannica.com/nobel/cap/opolchr001a4.html


How theHuman Genomewas Sequenced:

(See Text)

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?????????????? ?


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?????????????? ?


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


T

TA

ATACTGGAGC


TC

TA

ATACTGGAGC

ATGACCTCG GGCAAAGCCTCG ?

TCC

TA

ATACTGGAGC


TCCG

TA

ATACTGGAGC


TCCGTT

TA

ATACTGGAGC


TCC TTTCG G

TA

ATACTGGAGC


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


ATACTGGAGC TCC TTTCG GGAA

ATACTGGAGC TCC TTTCG

ATACTGGAGC TCC TTG



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 GG


G CATACTGGA C TC TTTG

ATACTGGAGC TCC TTG


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 GG


G CATACTGGA C TC TTTG

ATACTGGAGC TCC TTG


ATGCCTGAAATGC

CGTTACGTGATGATGCC

AATGCGTCATG

(a) Unaligned segments:

ATGCCTGAAATGC

CGTTACGTGATGATGCC AATGCGTCATG

(b) Aligned segments:

8. Use computer algorithms to align segments.

Polymorphisms. Monomorphism: Section of DNA where the nucleotide sequence is the same for everyone in the population. Polymorphism: Section of DNA with.

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