Lab 10' Allelotyping II

1
Lab 10. Allelotyping II Blood clotting SNPs
by PCR-SSP
Four targets FactorV Prothrombin CD28 CRP
(C-Reactive Protein) (control)
2
Method of SNP Detection

• Sequencing (Lab 9 P53 gene)
• PCR-SSP (Lab 10Blood Clotting Factors)
• (SSP) Sequence Specific Primer

Wild-Type FactorV Antisense GATGAACCCACAGAAAATGA
TGCCCA FactorV-G-sense CAAGGACAAAATACCTGTATTCCTC
gln Mutant FactorV-A-sense
CAAGGACAAAATACCTGTATTCCTT arg
3
Proth Antisense TCTAGAAACAGTTGCCTGGCAG Proth-G
Sense TGGGAGCATTGAGGCTC Proth-A Sense
ACTGGGAGCATTGAGGCTT WT CD28-T
Antisense ATTTTCTGGGTAAGAGAAGCAGCACT CD28
Sense ACCTACTCAATGCCTTCTGGAAATC Mutant CD28-
C Antisense ATTTTCTGGGTAAGAGAAGCAGCACC
4
CD28 (2q33) binds T-cell surface antigen-ligands
B7-1 (CD80) and B7-2 (CD86 Cause production
cytokines (interleukin-2 (IL-2)) Leads to an
increased cell proliferation. CTLA4 interaction
with the same ligands downregulates this
response. CD28 and CTLA4 molecules regulate
the immune responses to self- and non-self
antigens by controlling antigen-specific T-cell
activation.
Chromosome 2
5
Factor V Leiden Mutation (1q23) promotes
continued thrombin generation and a
hypercoagulable state (insensitive to
inactivation) Most common hereditary blood
coagualtion disorder in U.S. 5 of the in the
Caucasians 1.2 of the African Americans Heterozy
gotes Increased risk venous thrombosis 3-8 fold
Homozygotes Increased risk venous thrombosis
30-140 fold
Chromosome 1
6
Factor II (prothrombin G20210A) mutation
(11p11-q12) in the 3-untranslated region at base
20210, near Poly-A cleavage site Second most
common genetic defect for inherited
thrombosis Causes elevated plasma prothrombin
levels, more thrombin, and gt risk for thrombotic
via excessive growth of fibrin clots
Chromosome 11
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Use DNA from lab 9 (whole blood for P53) Prepare
amplifications Tue Amplify Read Thursday via AGE
Expected Results (for each target)
compare to CRP amplification control in each case