IDENTIFICATION OF POLYMORPHIC ALLELES

14.04.09

Quiz

If you are to prepare a %3 agarose If you are to prepare a %3 agarose gel, what should be the amount of gel, what should be the amount of agarose in the 75 ml 1xTAE buffer? agarose in the 75 ml 1xTAE buffer? Show your calculations.Show your calculations.

If you cross a heterozygous If you cross a heterozygous wildtype with an ebony mutant wildtype with an ebony mutant what will be the phenotypic ratio of what will be the phenotypic ratio of your F1 generation?your F1 generation?

Quiz

If you are to prepare a %3 agarose If you are to prepare a %3 agarose gel, what should be the amount of gel, what should be the amount of agarose in the 75 ml 1xTAE buffer? agarose in the 75 ml 1xTAE buffer? Show your calculations.Show your calculations.

In 75 ml for %1 percent: 0,75 gr %3 percent: 2,25 gr

ee ++

e e eeee +e+e

ee eeee +e+e

1:1 ebony: wildtype

Answer:Answer:

ee ++

e e eeee +e+e

++ +e+e ++++

F1 generation ++ ++

e e +e+e +e+e

ee +e+e +e+e

F2 generation

Phenotype ratio3:1, wild-type:ebonyGenotype ratio 1:2:1, homozygot WT, heterozygot WT, homozygot ebony

Today’s experiments: You will work as 4 groups First prepare 2% agarose gel1. Weigh 0,8 gr agarose into a flask2. Put 40ml 1X TBE buffer3. Dissolve the agarose completely by heating in the

microwave4. When boils, remove the flask from the microwave 5. Waits until it cools to 55C6. Add 2.5µl EtBr and mix7. Pour the melted agarose into gel apparatus8. Let it to harden9. Mix 5 µl of loading dye+ 5 µl of DNA sample, load on

agarose gels10. Run agarose gels at 150 V for 15 min.11. Observe under UV light

Electropherosis:

A method to seperate, identify and purify DNA fragments

2 types of gels:1. Agarose gels2. Polyacrylamide gels

Agarose Gels:

From sea weed Cheap, non—toxic Low resolving power, but a higher range

(200bp-50kb) Run in horizantal configuration In order to observe, EtBr is used EtBr intercalates DNA and it flouresces

under UV light, so we can detect the location of the DNA fragments on the gel

Agarose Gels:

Agarose gel electrophoresis unit

Agarose Gels:

Polyacrylamide gels:

Highly toxic, synthetic chemicals Prepared with acrylamide and

bisacrylamide. In the presence of free radicals, it

polymerizes into long chains

Polyacrylamide gels:

By changing acrylamide and bisacrylamide ratio, you can change the size of the pores

Polyacrylamide gels:

High resolution power, but a shorter range

(5bp-500bp) Vertical configuration

Polyacrylamide gels:

Polyacrylamide gels:

In order to visualize DNA, silver staining method can be used

Ag+ ions bind to (-)ly charged DNA, Reduced to Ag which has a brown color

Polymorphisms:

Common variation in DNA sequence It is a kind of variation related to biodiversity,

genetic variation, and adaptation Presence of more than one genetically

distinct type in a single population Useful tools in genetic studies for linkage

analysis, prenatal diagnosis, criminal cases and paternity tests

1. RFLP (restriction fragment length polymorhism)

2. VNTR (variable number of tandem repeats)

RFLP:

Restriction enzymes can recognize and cut specific DNA sequences

Ex: Msp I enzyme can recognize CCGG Cfo I enzyme can recognize GCGC EcoR I can recognize GAATTC

RFLP:

-/- +/- +/+

RFLP:

VNTR: (variable number of tandem repeats)

Can be found on many chromosomes, and often show variations in length between individuals

Each variant acts as an inherited allele, can be used for personal or parental identification

Their analysis is useful in genetics and biology research, forensics, and DNA fingerprinting.

Today’s experiments: You will work as 4 groups First prepare 2% agarose gel1. Weigh 0,8 gr agarose into a flask2. Put 40ml 1X TBE buffer3. Dissolve the agarose completely by heating in the

microwave4. When boils, remove the flask from the microwave 5. Waits until it cools to 55C6. Add 2.5µl EtBr and mix7. Pour the melted agarose into gel apparatus8. Let it to harden9. Mix 5 µl of loading dye+ 5 µl of DNA sample, load on

agarose gels10. Run agarose gels at 150 V for 15 min.11. Observe under UV light

Today’s experiments: You will make a paternity test1) On each bench, you have 5 DNA

samples: mother, child and 3 father candidates

2) We will identify the father by checking 2 polymorhisms on different chromosomes 1. group RFLP (on chromosome 2)2. group VNTR (on chromosome 5)

Today’s experiments: 1. group RFLP (on chromosome 2) DNA fragment includes a Single nucleotide

polymorhism (G or T) Msp I enzyme CCGG

600bpCCGG CCTG

Digest with Msp I

400bp 200bp 600bp

Today’s experiments: On agarose gel: load 3 µl 100bp marker

bp

bp

bp

mother child father3 father1 father2marker

-/- +/- +/+ +/- -/-100

200

300

400

500

600

Today’s experiments: 2. group VNTR (on chromosome 5) DNA fragment includes different number of

(CA) repeats You dont need to load marker

Expected results:

RFLP: 1. group:

616bp

400bp

200bp

mother child father3 father1 father2

marker

-/- +/- -/- +/+ +/- 100

200

300

400

500

600700800

VNTR: 2. group:

mother child father3 father1 father2

marker

2/4 1/2 2/6 1/2 1/3

100

200

300

400

500

600

4

3

2

1

800

700

Monohybrid cross results Ebony vs. wildtype

Hypothesis:Hypothesis:

Body color is an autosomal trait and Body color is an autosomal trait and ebony type is recessive to the wild ebony type is recessive to the wild type”type”

To test the accuracy of our To test the accuracy of our hypothesis, we need to calculated hypothesis, we need to calculated to which extent our observed to which extent our observed results are departed from the results are departed from the expected resultsexpected results

ee ++

e e eeee +e+e

++ +e+e ++++

F1 generation++ ++

e e +e+e +e+e

ee +e+e +e+e

F2 generation

Phenotype ratio3:1, wild-type:ebonyGenotype ratio 1:2:1, homozygote WT, heterozygote WT, homozygote ebony

Count Ebony vs. Wildtype Drosophila