13. Molecular Techniques and Diagnosis - Analysis of DNA at the Gene Level Flashcards
What does DNA gel electrophoresis allow us to see?
The colourless DNA fragments that have been cut up.
Which direction do DNA fragments travel in in gel electrophoresis?
Towards the positive electrode.
What are the four requirements of gel electrophoresis?
Gel (matrix that allows separation of fragments), buffer (allows charge on DNA sample across gel), power supply (generates charge difference across gel) and stain/ detection (identifies presence of separated DNA).
How are DNA fragments separated in gel electrophoresis?
By size and charge.
Why use restriction analysis?
To investigate size of DNA fragments, mutation, DNA variation and to clone DNA.
What are plasmids?
Small circular double stranded DNA found in bacteria.
How are genes cloned?
The DNA of interest and plasmid vector are joined to form a recombinant DNA molecules which is introduced into a cell using a vector then the cells into bacterium. The DNA fragments will have resistance to the bacteria they’re grown in so only the cells that reproduce and survive in the bacterium contain the new gene.
Why clone human genes?
To make useful proteins (like insulin), find out what genes do, genetic screening and possibly gene therapy in the future.
How is proinsulin synthesised by bacteria?
Mammalian proinsulin mRNA is taken and undergoes reverse transcriptase to form proinsulin cDNA. The proinsulin cDNA is joined to a plasmid to form a recombinant plasmid which is used to infect E. zocalo cells.
When was PCR discovered and by who?
1983 by Kary Mullis.
How does PCR work?
Heat to 95 C so the DNA denatures. Then cool to room temperature so DNA renatures and the forward and reverse primer can join.
Why use PCR?
To amplify specific DNA fragments, to investigate single base mutations, to investigate small deletion of insertion and to investigate variation in genetic relationships.
What do endonucleases do?
Recognise and degrade foreign DNA. They recognise and cut specific DNA sequences, which are usually palindromes.
