Using Gene Sequencing Flashcards
What is a genome
Total of all genetic material in an organism
What is PCR
A technique for the amplification of DNA outside of the body
How does PCR work
DNA is heated to 95 to break hydrogen bonds and separate strands
Mixture is cooled to 50-65 to allow primers to anneal at 3’ end
Heated to 72 for DNA polymerase (taq) to attach nucleotides to replicate the region of DNA
Repeat this to amplify DNA
What 3 steps can PCR be summarised into
Denaturation
Annealing
Extension
How do we sequence DNA
DNA is chopped into smaller pieces
Double strands are separated into single strands
PCR is used to amplify the DNA
Terminator bases are added to the strands
The tags allow bases to be read rapidly
What are terminator bases
Modified versions of bases that have a fluorescent tag attached
It stops anymore bases from being added
What are 2 ways of using sequencing information
Predicting amino acid sequences - help us predict which genes code for which proteins and how the amino acids might join together in a polypeptide chain
Disease management - looking at mutations which cause disease. We can identify the mutations and then decipher how the mutation in the protein causes the symptoms
What is DNA profiling
Identifying the small differences (generally found in the introns) in our DNA that allows us to make an individuals DNA profile
What are micro and mini satellites
Short sequences of DNA which are repeated many times within the introns
Mini - 10 to 100 base sequences repeated 50 to several hundred times
Micro - 2 to 6 bases repeated 5 to 100 times
Gel electrophoresis
DNA fragments are placed in wells in an agarose gel medium in a buffering solution
The gel contains a dye that binds to the DNA fragments in the gel
The dye will fluoresce when placed under UV revealing a pattern of DNA
An electric current is passed through the DNA fragments moving towards the positive anode due to a negative charge on the phosphate backbone
Southern blotting
What is southern blotting
An alkaline buffer solution is added to the gel after electrophoresis and nylon filter paper is placed over it
This draws the solution containing the DNA fragments from the gel to the paper
The alkaline solutions also denatures the DNA fragments so the strands separate and the base sequences are exposed
Gene probes are then added to the filter after this process to bind with the complementary DNA strands in a process called DNA hybridisation
When is DNA profiling used
In forensic science
In paternity testing
