Molecular biology 3 Flashcards
What method do we use when sequencing DNA?
Examination of DNA sequences/fragments
via Direct (Sanger) sequencing, also called the
“chain termination method”
Each peak corresponds to one nucleotide
each column represents the type of nucleotide
How does direct sanger sequencing work?
compare PCR and sanger
This is a very similar process to PCR
1. Template DNA
2. Forward and reverse primers
3. dNTPs (deoxyribonucleotides)
4. DNA polymerase
5. Buffers and catalysts
sanger
1.Template DNA
2. Forward primer (depends on the direction you want to sequence)
3. dNTPs (deoxyribonucleotides)
4. DNA polymerase
5. Buffers and catalysts
6.ddNTPs (dideoxyribonucleotides) (in structure lacks two oxygens so 2 H’s at the bottom instead.
this means that the phosphate cannot react as it needs oxygen so no new nucleotides can join
How do nucleotides join?
phosphate reacts with the oxygen at the 3’
direction = 5’ to 3’
How does sanger sequencing work?
Sanger sequencing results in the formation of extension products of various lengths terminated with dideoxynucleotides at the 3′ end. The extension products are then separated by Capillary Electrophoresis or CE. The molecules are injected by an electrical current into a long glass capillary filled with a gel polymer.
The results are then shown as a chromatogram
When there is an N that means that the position wasn’t probably sequenced.
What are haplotypes?
A group of alleles in an organism that are inherited together from a single parent.
Name other DNA sequencing techniques?
Microsatellites are small pieces of DNA that repeat. They are usually only a few of the DNA base pair letters in size. The combination of letters and the patterns in which they repeat can be unique between individuals, which is like fingerprints.
AFLPs (Amplified Fragment Length Polymorphisms)(normally used for plants)
highly polymorphic so lets you distinguish between different things.
It is a PCR-based technique that uses selective amplification of a subset of digested DNA fragments to generate and compare unique fingerprints for genomes of interest.
Single Nucleotide Polymorphisms (SNPs). They can act as biological markers, helping scientists locate genes that are associated with disease
What are the advantages and disadvantages of microsatellites?
advantages
-easy to run
-easy to share
-easy to score
-easy to quantify and scale up
Disadvantages
-if there are any mutations it will not have the right microsatellites
-It is sensitive to if polymerase misses some of the repeats
Describe high-throughput/next generation sequencing (NGS)?
technologies that sequence DNA and RNA in a rapid and cost-effective manner. It is used to determine the order of nucleotides in entire genomes or targeted regions of DNA or RNA. Even sequences are short you still get a lot of data
