5.5 Flashcards
What is DNA sequencing
process of determining the sequence of nucleotides in a piece of DNA. the scale of DNA sequencing can vary from a singular gene to a whole genome
what is PCR
A polymerase chain reaction is a technique used to amplify DNA to make lots of copies of a specific region of DNA. Necassary 4 testing, analysing or to use a DNA sample
what process is PCR very similar to
DNA replication
where would DNA samples come from?
Crime scenes, or person being tested for a genetic disease
What is the first step of PCR
- Denaturation
- PCR reaction mixture is heated to 95C
- DNA is denatured i.e, separates to form 2 single strands
- high temperature is the cause of the separation of both the template strand n the coding strand
what is the 2nd step of PCR
Annealing -
- PCR reaction mixture is cooled to 55C
- Allows primers to anneal to the template DNA - acts similar to mRNA coding against the template strand
what is the 3rd step of PCR
Extension, PCR reaction mixture is heated to 72C, And DNA polymerase moves down the template, synthesising new DNA
Step 4 of PCR and the resultant of the whole PCR
denature the PCR once again to 95C. this separates the DNA and stops DNA synthesis.
IT then redoes this process over and over again replicating the DNA segment over n over again
How is PCR integrated into DNA sequencing
- DNA sample received
- external DNA from sample
- Amplifying DNA- make more copies
Shared aspect w PCR^
What occurs after DNA is replicated in PCR in the process of DNA sequencing
sanger sequencing reaction which separately identifiees the position of each nucleotide.
- using 4 special PCRs = normal PCRs w a chain-terminating nucleotide
what occurs to a nucleotide in the steos of DNA sequencing
A normal DNA strand has a bond to its hook but a chain-terminating nucleotide hence theres no bond
what does the chain terminating nucleotide do
Dna polymerase will continue adding upon nucvleotides up untul the chain terminating nucleotide, stopping the sequence to contineu
how is the length of a dna sequence determined
run all 4 PCR reactions on an electrophoresis gel, to determine the lengths of the fragments in each reactions
Larger - DNA pol stops late
small - dna stops slowly
what r the three rules for determining a DNA sequence
- work from smallest- largest frament
- Identifying which PCR reaction the fragment came from
- The next nucleotide is compulsory to the chain termianting nucleotide
what is gel electropherosis
