For HIGH res sequencing Perform PCR on fragment to be sequenced as usual for
You terminate the sequence at different base pair lengths using the terminating dideoxynucleotides
In the GEL, you have four wells each with 4 PCRs - one PCR system has ddATP, ddTTP, ddCTP, ddGTP
Gel Electrophoreesis Results
Predict results given the target gene sequence ***** esp helpful studying for the midterm
Dideoxynucleotides
Separate DNA polymerase from the DNA strand - terminates synthesis
Electrophoresis and BP Resolutions
EP can separate different DNA fragments based on length of the fragment
Medium Resolution
rimers plus restriction enzymes
High
When you need to know every single base pair in the sequence - like for insertion and substitution
Low Resolution
Primers Only Simplest form , you are looking at large insertions or deletions, not single bp mutations -Primers used to isolate DNA
PCR Primers
PCR Primers alone are sufficient to separate DNA fragments representing specific polymorphisms
Separating Fagments
Standard "kitchen" electrophoresis is suffient to separate fragments
Required Resolution
Required Resolution is typically on the order of 100 bp - spacing might not be uniform but about 100
ALU Jumping Gene
Part of gene that is benign, has no real function right now. Just replicates itself and reinserts itself into other parts of the genome. Only appears in primates. About 1 million copies in the genome. Two possible polymorphisms (one with ALU insertion, one without) Insertion is about 300 bp long.
IF you have the gene, it is 300 bp longer IF you don't, you have 300 shorter.
Can Cause Disease
If it is inserted into a malignant part, you can get diseases
Variation 1
You can use one PCR -use fluorescent labeling
PCR Primers
Primers alone not sufficient to separate DNA fragments representing specific polymorphisms (the mutation is too small to detect with just primers)
Restriction Enzymes
Cut the gene into fragments representing specific polymorphisms
Require resolution
Typically on the order of 10-20 bp (but this can be misleading) -you have to see if a restriction enzyme exists that can detect your polymorphism
Example: Sickle Cell Anemia
Single Substitution Mutation -There is a RE that can cut the gene at exactly the sequence when there is no sickle cell anemia -When you have sickle cell the RE does not cut the DNA -Through EP, we can see if there is a cut in the DNA (no sickle cell) or no cut (sickle cell anemia)
Primers and RE
Can isolate small gene segments
Polymorphims
May differ only by a few BP (SNP) without associated restriction enzyme
Required Resolution
ONE BP need to identify everything
Details/Structure
Phosphate groups cannt attach because no hydroxyl (OH) groups - chain terminates
For midterm
know which end i 5' and 3' be able to draw sequence using a given sequence
Variation 2
Use an Automatec DNA Sequencer (get bunch of colored waves that correspond to certain wavelengths for the nucleotides)
Detects Genotypic Errors
Technology to identify genetic polymorphisms or mutations look at known gene sequences - diagnosis not discovery