Types of Polymorphisms I. Protein/enzyme polymorphisms Blood groups II. DNA Polymorphisms 1.Single Nucleotide Polymorphisms (SNP) 2.Tandem Repeat Polymorphisms

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...


Deletion

Deletion Polymorphism

Inversion Polymorphism


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


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.

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

Tools in Molecular Genetics:1. Electrophoresis2. Probes3. Polymerase Chain Reaction4. Restriction Enzyme5. Dideoxy Nucleotides6. 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) 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)


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


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


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

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.

http://www.sanger.ac.uk/genetics/exon/

Finding a SNP

http://www.wtcrf.ed.ac.uk/genetics/images/snp1.jpg

SNP Genotyping Machine(7000 genotypes per day)

Wellcome Trust Clinical Research Facilities

Potential New Method:Nanopores

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) 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?????????????? ?


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)

ATGCCTGAAATGC

CGTTACGTGATGATGCC AATGCGTCATG

(b)

Documents

Types of Polymorphisms I. Protein/enzyme polymorphisms Blood groups II. DNA Polymorphisms 1.Single Nucleotide Polymorphisms (SNP) 2.Tandem Repeat Polymorphisms