Manipulating genomes Flashcards
Steps of PCR
-Requires DNA, free nucleotides, Taq DNA polymerase + two primers
-Heat DNA to 94-96’C to denature DNA/ hydrogen bonds break so single stranded
-Cool to 50-65’C- primers anneal to 3” ends
-Heat to 72’C- DNA polymerase attaches to nucleotides, binds nucleotides to make new strand
Why PCR?
-To amplify DNA for DNA sequencing, profiling, genetic modification etc
Applications of PCR
-Forensic science
-Detecting mutations
-Oncogenes
-Tissue typing-donor and recipient tissues typed to decrease risk of rejection
Why Taq DNA polymerase?
-Will not denature at 94-96’C due to being from thermophillic organism, increases rate of DNA replication
Steps of DNA profiling
-DNA obtained from cheek swab etc
-PCR
-Digested with restriction enzymes (restriction endonuclease)- cut at specific recognition sites- makes fragments of varying lengths
-Electrophoresis
-Hybridisation- gene probe (florescent or radioative) anneals to DNA to make it visible
-Development- alkaline solution added to gel plate, nylon filter placed over it (southern blotting)- denatures and make it single stranded
-observe banding patterns
What part of DNA is being observed in DNA profiling
The Variable Number Tandem repeats (non coding DNA (introns) unique to each person. (different number of repeats and lengths)
Definition of DNA sequencing
-The process of determining the base nucleotide sequence of DNA
Applications of PCR
-forensic science
-maternity/paternity disputes
-analysing genetic disease
Steps of Sanger sequencing (first method of DNA sequencing)
-Extract DNA, cut into fragments using restriction enzymes, degrade into single strands
-Amplify by PCR
-4 seperate containers, dideoxynucleotides of A,T,C,G + free floating nucleotides
-Dideoxynucleotides bind randomly to sequence when read by DNA polymerase, makes fragments of different lengths
-Electrophoresis. Then bases can be read by the radioactive label on the dideoxynucleotides on x-ray
-Sections placed in order by matching overlapping regions to read base sequence
Applications of DNA sequencing
-Human Genome Project
-Allows us to compare genomes of individuals and species to identify evolutionary relationships, analyse alleles and understand diseases. Understand epigenetics
-Predicting animo acid sequences
-Synthetic biology (production of medicine, info storage on DNA, nano tech, biosensors etc)
Brief overview of Pyro/throughput sequencing
-Sequencing by synthesis of DNA, one base at a time, read by the light emissions when bases added
What is bioinformatics?
-Storage of large amounts of universal DNA and proteins
-Allows comparison of genome and animo acid sequences
-Used in computational biology to model protein structures from DNA sequences
