Testing for Familial Hypercholesterolemia

Caused by a Mutation in the LDLR Gene

A simulation of a genetic test using Gel Electrophoresis

Nature vs. NurtureNature = genes and their effects

Nurture = environmental influences

ExamplesSickle Cell Disease is heavily nature because it is related

to a specific gene that are inherited Hypercholesterolemia is a blend of Nature and nurture

Nature = genes such as APOB, PCSK9, LDLRAP1 and LDLR

Nurture = diet, exerciseMost traits are a combination of Nature and NurtureMost traits are controlled by many genesMost traits are effected my several environmental

influencers

Human GenomeWe have two in every cell!

Maternal and Paternal sources

Human Genome Complete set of human DNA About 3 billion base pairs Segmented into 23 different

sections called chromosomes23(egg) + 23(sperm) =

46(zygote) We have two of every gene.

Genes come in various forms.Ex: ‘wild type’ hemoglobin

and sickle cell hemoglobin

HypercholesterolemiaHigh cholesterol

High cholesterol leads to Coronary Artery Disease (CAD) which often results in heart attacks.

Our Lab This lab tests one of the genes (LDLR) associated with high

cholesterol Positive result means an 85% risk of heart attack for men in

there 40s and 50s or women in 50s or 60s Negative result means average risk of heart attack

Due to environmental factors (diet, exercise)

Background: Parents of Sylvia (2 yr old) both have high cholesterol. They want to know if it is related to their genes and if they passed the genes to their daughter. DNA samples have been collected from Mom, Dad and

Sylvia The LDLR gene in each of the samples have been amplified

and digested with a restriction enzyme. We will perform the gel electrophoresis procedure, analyze

the resulting gels and inform the parents of the outcome of the test and their daughters risk level of developing high cholesterol.

Day 1How to use a micropipette

Look at the number on the top of your micropipette

Look at the numbers on the side (through the window)

Push down the top “plunger” - feel first and second stopsFirst stop is for picking upSecond stop is for dispensing

Watch how to pick up a tip

Day 1How to use a micropipette

Watch how to pick up a liquidAlways hold the liquid in one hand while picking it

upONLY go to the first stop when picking up a liquid

Watch how to dispense a liquidAlways go to the first stop, then slowly to the

second stopPull micropipette out BEFORE releasing the

plungerWatch the technique

Day 1 - Practice Loading

ALWAYS use a TIP with your micropipette

ALWAYS keep the micropipette VERTICAL when you have liquid

1 l (micro) = 10-3 mL (milli) = 10-6 L

When picking up liquid, push to the FIRST stop only!

When dispensing liquid, push to the FIRST stop, then the SECOND stop

When dispending liquid, ALWAYS raise the tip out of the liquid before letting go of the button!

Day 2 - Loading/Running Gels

Only two group members at the gel at any given point

Each group member should load at least one sample

Load 35 L sample into the wells (label the wells on the diagram so you know where every sample is!) Tap the sample so that all is at the bottom of the container

Let me know once all samples are loaded

Work on the ‘paper lab’ activaty while you wait

Day 2 DNA samples Sample A – A known DNA sample called a “Standard” that

contains fragments of known length

Sample B – Control DNA that is known to have two normal copies of the LDLR gene

Sample C – Control DNA that is known to have two mutant copies of the LDLR gene

Sample D – Sylvia’s mom’s DNA

Sample E – Sylvia’s DNA

Sample F – Sylvia’s dad’s DNA

GroupsGroup 1 Group 2 Group 3 Group 4 Group 5 Group 6

Olivia Shanice Oliver Emma Liam

Patrick Suzanna Ashley Clara Alana

Jessica Marina Ethan Mei Yarden

Charles Jack Becca Mike

Day 3: Gel AnalysisPlace your gel on the light box

Do a detailed sketch of your Gel and the reference gel in your packet

Include in the sketch # and position of bands Band brightness Label the lanes

Use both gels when drawing conclusions about Sylvia.

Only one group at the light box at one time.