Molecular Diagnosis Flashcards
What kind of sequences do the restriction nucleases recognise and what are they?
Palindrome sequences, they read the same forwards as backwards.
How many base pairs do the restriction enzymes usually recognise?
4,6,8 usually the even number
What does the cutting of the DNA sequence leave and what happens to them?
Leaves sticky ends that can either be rejoined or another complementary fragment is attached by DNA ligase.
What does DNA ligase do?
Creates a phosphodiester bond to join two fragments of DNA together.
What does restriction analysis mean we can do?
Isolate fragments of DNA by targetting specific areas based on the restriction sites of the restriction endonucleases.
What can this along with DNA electrophoresis be used to investigate?
Investigate size of DNA fragment (see deletions) Investigate mutations (sickle cell disease) Investigate DNA variation (genetic finger printing) Gene cloning
What is used for DNA cloning, what are its features and what is it used as?
Plasmid - mini chromosome, small circular DNA, can transfer to other bacteria, carry genes to replicate independently, can contain antibiotic genes.
A vector
Why carry out gene cloning?
Make useful proteins (eg insulin), find out what genes do, genetic screening (huntingtons, CF, BRCA1/2 gene, gene therapy (CF)
What is the process of gene cloning?
Plasmid is cut using restriction enzymes, gene of interest is added to create recombinant DNA molecule. This is introduced to bacterium via transformation (they are the suitable host cells where replication will take place). Bacteria containing recombinant DNA are place in an environment to multiply. Clone containing gene of interest then identified and isolated.
What does the plasmid also usually contain and why?
An antibacterial resistance gene, in order to positively select for the bacteria that have taken the plasmid up.
How is insulin cloned?
You take the human insulin gene as mRNA as this codes for the protein. You use reverse transcriptase to convert the mRNA back into cDNA and join the fragment to the plasmid so it can be put into the bacteria or mammalian cells for replication of the insulin gene.
Which cell is better for the insulin gene to be placed in for replication? why?
Mammalian cells as they contain all of the post-transcriptase systems to allow the formation of mature insulin.
What is DNA gel electrophoresis used for?
To separate different sized DNA fragments.
What is the process of gel electrophoresis?
A solution of different fragments is placed in a well at the negative anode end. A charge of the anodes encourages the negatively charged DNA fragments to move towards the positive anode. Larger fragments are slowest and move the least. Fragments of known size are used as a reference.
What does gel electrophoresis (DNA and protein) require?
Gel, buffer (of salt etc to allow the charge to be carried across the gel), power supply and stain (because DNA and proteins are usually colourless).
What do we use PCR for?
To amplify a sample of DNA for testing
How does PCR work?
It uses repeated copying of the target DNA using a pair of primers that uniquely define the region to be copied and thermostable DNA polymerase (TAQ) which can bind once the primers are bound. It involves fluctuation in temperature to denature, anneal and resynthesise the DNA. This amplifies the DNA exponentially.
What is a benefit of using thermostable DNA polymerase in PCR?
You dont have to keep replacing the enzyme as it wont denature at high temperatures.
What are the primers in PCR?
Small molecules of complementary DNA that (once the temperature has reduced a little after heating to split the strands) has preferential binding to the strands of DNA (over the strands themselves) and forms the site where the thermostable DNA polymerase can bind.
