MGD Session 7 Molecular Diagnosis 1+2 Flashcards
DNA Sequencing What is the difference between ddNTP’s and dNTP’s?
ddNTP’s are a variation of dNTPs lacking a 3’ OH group, so polymerisation cannot occur
DNA sequencing
What does the structure of ddNTP mean for the DNA strands it is added to?
Strand terminates
DNA sequencing Why does the use of ddNTP produce lots of new fragments at different lengths?
Strands terminate at different points depending on where ddNTP added at each part.
How many test tubes used in DNA sequencing and why
Four, each with of type of ddNTP in (ddATP, ddGTP, ddCTP, ddTTP)
DNA sequencing How can you measure size of strands produced?
Gel electrophoresis
Why do DNA strands need to be denatured in gel electrophoresis?
So they have charge
Explain process of DNA electrophoresis.
Adequately.
Lots of fragments of different sizes produced. Can all be electrophoresised to separate according to size. Position each test tube fragment comes in indicates sequence, as each of four wells is different nucleic acid.
What are restriction endonucleases?
Bacterial enzymes that recognise and cut specific DNA sequences known as restriction sites
What word can be used to describe the structure of many restriction sites, and how long are they?
Palindromes, 4-8 bp
How do bacteria protect their own DNA from restriction endonucleases?
Methylation
What does the cutting of a DNA sequence by restriction endonuclease leave?
Sticky ends
How can sticky ends be joined?
Via DNA ligase
What four things can be found out by combining electrophoresis and Restriction endonucleases?
Size Huge mutant variety of clones
Mutations
DNA variation
Gene cloning
Gene cloning
What is a plasmid?
Small circular ring of DNA
What property of a plasmid makes it a good vector?
Can transfer from bacteria to bacteria
Via what method can you add a desired gene into a plasmid?
Use of restriction endonucleases on both the target gene and the plasmid. Complementary sticky ends will be produced, which will combine under influence of DNA ligase.
What is the process called by which a modified plasmid is introduced into a bacteria
Transformation
Why is it useful for a plasmid to take up an antibiotic resistant gene as well as a desired gene?
Means those bacteria who didn’t take up plasmid will be killed by specified antibiotic.
Give three reasons for cloning genes
Useful proteins, find out what genes do, screen for bad genes
Name a process of DNA analysis which can be used to work out structure of DNA at nucleotide level
DNA sequencing
Name three methods of assesing DNA at the gene level
Name two ways of assesing DNA at chromosome level
Karyotyping
FISH
Name three ways in which proteins can be analysed
Protein electrophoresis
Immunoassays
Enzyme assays
What is Gel electrophoresis used for?
to separate different sized DNA fragments.
Where are the solution of different fragments place in gel electrophoresis?
In a well at the negative annode end
What charge does DNA have, and why?
Negative, as acidic and has lost hydrogens
Which way does DNA move in GE (gel electrophoresis)
Towards the positive anode
Which move faster in GE, large or small fragments?
Small
How do we know the size of DNA/Protein during GE by the distance it goes?
By using a reference sample of fragments of known size
Name four things required by Gel electrophoresis and why
Gel – A matrix that allows separation of DNA fragments
Buffer – Allows charge on DNA samples across the gel
Power Supply – Generates charge difference across the gel
Stain – To identify the presence of the separated DNA
What does PCR do?
amplifies DNA segments by repeated copying of the target DNA
What is the name of the DNA polymerase used in PCR, and why is it used?
TAQ DNA polymerase, used as it can survive high temps of 96* needed to break DNA double bonds
What do the pair of primers in PCR do?
Uniquely define region of DNA to be copied
Give three stages of PCR and associated temperatures
- Denaturation at high temperature (94 – 960C)
- Renaturation (annealing) at lower temperature (50 - 650C)
- DNA synthesis at medium temperature (75 - 800C)
Give three investigations that can be provided via PCR
What is the difference between protein and DNA gel electrophoresis?
Proteins move towards either annode or cathode, so separated by charge
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