Gene Sequencing Flashcards
What is PCR?
Polymerase Chain Reaction
-A technique for amplification of DNA in vitro
What is the process of PCR?
-A sample of DNA is taken and undergoes PCR
-The DNA is heated to 95 degrees causing the hydrogen bonds to break and separate the two strands of DNA (denaturation)
-The mixture is then cooled to 55 degrees allowing the primers to anneal to the 3’ end of each strand (annealing)
-It is then heated again to 72 degrees allowing Taq polymerase to attach nucleotides
-The process is then repeated several times to amplify the DNA (extension)
Why is Taq polymerase used?
It is heat resistant as it is taken from bacteria living in thermal hot springs
What is a genome?
The entire genetic material in an organism
Who invented PCR?
Kary Mullis in 1993
What is DNA sequencing?
Identifying the base sequence of a DNA fragment
What is the process of DNA sequencing?
-DNA is chopped into smaller pieces and double strands are separated into single strands
-PCR is used to amplify DNA
-A primer is added
-The fragments are added to four test tubes (A, T, G, C) each containing nucleotides and a polymerase enzyme
-The fragments grow until a terminator base stops them
-Chains are denatured and put in gel electrophoresis
What is a terminator base?
Modified versions of bases with a florescent tag attached which stops anymore bases from being added
What happens when DNA has been sequenced?
The raw data is fed into a computer system that reassembles the genome by looking at overlap between fragments
How can we use DNA sequencing?
-Predicting amino acid sequences to help predict which genes code for which proteins and how the amino acids may join in a polypeptide chain
-Disease management by looking at the mutations which cause disease and can see which mutation in the protein causes symptoms
What is the process of DNA profiling?
-DNA sample is cloned using PCR
-DNA polymerase and free nucleotides are added
-DNA is heated to break the hydrogen bonds
-DNA is cooled and primers are added
-DNA polymerase joins free nucleotides together
-Cycle is repeated to clone multiple copies of DNA
-Cloned DNA is cut into fragments of different lengths using different restriction enzymes
-Fragments are placed into wells and gel electrophoresis is carried out
-DNA fragments separate according to size and move towards anode
-Position of fragments is compared with other samples obtained
-Matching bands can be used to identify crime suspects, give a medical diagnosis or for paternity cases
What is an allele?
Different versions of the same gene
-Everyone has the genes for hair, alleles cause different hair colours
