Download Edvotek Kit #116: Genetically Inherited Disease Detection Using Pre

Edvotek Kit #116: Genetically Inherited Disease Detection Using Pre-Cut DNA
Sickle Cell Data Chart CHECKLIST
Name:______________________________________________Date:______________
INTRO:
Hemoglobin, which is present in red blood cells, is the carrier of oxygen to cell in the body.
It is a protein made up of two alpha chains and two beta chains. The alpha chain gene is located on
the short arm of chromosome 16, while the beta-globin gene cluster is on the short arm of
chromosome 11.
Sickle Cell results from a Point Mutation on the short arm of chromosome 11. A single
nucleotide polymorphism (SNP) between an A to T results in a new amino acid in the sixth position
of the beta chain of hemoglobin in red blood cells. In normal hemoglobin (Hb A), glutamic acid
(Glu) is present. In the mutated form Valine (Val) is substituted because of the point mutation and
the variant form of hemoglobin results (Hb S).
In capillaries, CO2 is a byproduct of metabolism that enters red blood cells (RBC) and is
converted to carbonic acid. The acidic pH reduces the affinity of O2 binding to hemoglobin, resulting
in the release of O2 to cells. When CO2 is released from RBCs in the lungs there is an increase in pH
which favors the binding of O2 to hemoglobin. In individuals with Sickle Cell Anemia, the binding
and subsequent transport of oxygen is compromised. It also causes the RBC to have a sickle shape.
Gregor Mendel predicted that offspring inherited traits by receiving one allele for a trait from
each parent. Therefore, offspring receive 2 alleles for each trait. Alleles can be dominant or recessive.
A dominant trait will mask a recessive one. Homozygous trait: both alleles are the same (either both
dominant or both recessive). Heterozygous trait: one allele is dominant and one is recessive.
This kit uses pre-cut DNA. The restriction enzyme MstII will recognize a restriction site in
Please check off as you go.
Pipetting into a gel:
______1) Dial up designated volume: 15 or 20 µL
______2) Obtain sample tube and open.
______3) Using micropipettor, obtain microtip by pushing nose cone into proper-size tip.
______4) * Hold sample about eye leve. May hold micropipette @ 45o angle from vertical (so hand
contaminants won’t fall into tube) or use vertical entry.
______5) Push plunger to FIRST Stop (DO NOT go to SECOND STOP!). This must be done before
entering solution so you don’t produce bubbles of agitation.
______6) Position tip into upper part of solution.
______7) Draw fluid into tip by slowly releasing plunger.
______8) Close sample tube and return it to sample rack.
*Micropipet should always be in VERTICAL POSITION while it contains liquid so liquid does not
enter nose cone! ! !
Princeton Satellite Site
Spring 2010
K. McKone
Edvotek Kit #116: Genetically Inherited Disease Detection Using Pre-Cut DNA
Sickle Cell Data Chart CHECKLIST
______9) Pipet into gel by slowly pushing plunger to FIRST stop (DO NOT GO TO SECOND
STOP).
______10) KEEP plunger at FIRST stop and withdraw pipet from gel.
______11) Slowly release plunger and eject tip into waste beaker.
______12) If all samples are loaded, close box top and connect color coded leads to power supply.
______13) Turn on power supply and set Volts to ~ 100. Run for 20-25 minutes.
Sketch resulting bands below and write Hetero-, Homozygous-Dominant or
Recessive; Include phenotype: Normal, Carrier, or Hemophilia
Another diagram is provided if your samples are loaded DIFFERENTLY.
1
2
3
4
5
6
7
Sickle
Carrier
Normal
Mom’s
Child’s
Dad’s
Practice
cell gene sample
gene
DNA
DNA
DNA
sample
sample
___ µL
___ µL
___µL
___ µL
___µL
___µL
___µL
red
clear
blue
green
yellow
purple
Lane
1
2
3
4
5
___ µL
___ µL
___µL
___ µL
___µL
8
Practice
___ µL
Big
orange
Big
orange
6
7
8
___µL
___µL
___ µL
Samples
Volume
Loaded
Sample
Tube
Color
Band
Patterns
&
Phenotype
Based on the gel: What is the genotype of the child? Homo-Dominant, Hetero, or Homo-Rec
What is the phenotype of the child (circle one): Normal,
Princeton Satellite Site
Spring 2010
Carrier, or Sickle Cell
K. McKone