Flashcards in MCBG 13+14 - Molecular techniques Deck (47):
How does hybridisation work? 3
1.) Denature ds DNA
2.) Add a ssDNA or RNA probe (sequence of Nucleotides)
3.) Eithe probe is fluorescent/radioactive or you add a label
Why was it named southern blotting?
Discovered by Prof Sir Southern
What is a Southern/Northern Blot. One word
Hybridisation. (Gel electrophoresis + hybrisation)
How is western blot different from southern blot?
Its uses proteins
DOES NOT USE HYBRIDISATION
Describe Western blot
1. Gel Electrophoresis
2. Blotting onto surface
3. Adding labelling probs (antibodies) No hybrisation
Describe Southern Blot
1. Restriction enzymes cleave DNA
2. Gel Electrophoresis
3. Transfer to nylon
4. Hybridise with gene probe ( fluorescence or radioactive)
5. Detect labels
Sickle cell disease is a mutation in the HBB
gene. What is the most suitable way to get the
new-born’s HBB gene for a genetic test?
PCR (part of ) new-born’s HBB
After you PCR what can you then do after? 2/3
1. restriction analysis
2. gel electrophoresis (Southern blotting)
3. DNA sequencing
What is sickle-cell disease?
Whats the most common?
Group of blood disorders with the most common being sickle cell annemia.
MstII is a restriction enzyme; where are restriction
Whats a single-nucleotide polymorphism?
A different base. A specific base position in the human genome, the base C may appear in most individuals, but in a minority of individuals it doesnt
Name three diseases single-nucleotide polymorphism occur in?
Sickle Cell Disease
What units make up haemoglobin
HbA = a2b2
Alpha and beta subunits
Sickle Cell Anaemia. What mutation occurs?
Single Base mutation(exchange).
A->T in the B-globin gene
Glutamate -> Valine
Sickle Cell Anaemia. Why does the mutation effect RBCs?
Valine is hydrophobic. This distorts the shape of the cell. Causes it to collapse and form a sicle shape
Sickle Cell Anaemia. What is the name of the of Mutation (found on a biomed form) [hint aa]
What type of genetic disease is it?
Whats a mismatch mutation?
Whats a pyrimidine dimer?
Where two pyrimidines (C,U,T) bond
Whats an intercalating agent
molecules that may insert between bases in DNA, causing frameshift mutation during replication
Name sources of mutations
1. Ionising radiation
2. UV light
3. Oxidising chemicals
How is DNA read in transcription?
DNA is read 3’ to 5’
What direction is RNA synthesised in transcription?
mRNA synthesis 5’ to 3’
What direction is mRNA read translation?
mRNA is read 5’ to 3’
What direction is polypeptide chains synthesised?
polypeptide synthesis N to C
Single nucleotide changes can be split into two types.
Transition - Purine to purine
Transversion - (A/G-C/T)
Whats a missense mutations?
A mutation that causes any amino acid to be changed from its original
Whats a non-sense mutations?
A mutation that causes a premature stop codon
Whats a silent mutation?
A mutation that doesnt effect the final mutation
Whats a conservative mutation?
A mutation that changes an amino acid to one that has similar properties
Whats a non-conservative mutation?
A mutation that changes an amino acid to one that has different properties
What type of mutation is sickle cell anaemia? Sense missense or non-sense
Non-conservative MISSENSE Mutation
(a point mutation that doesnt cause a FS but changes the amino acid GLU-> VAL. So its a MISSENSE Mutation
Missense mutations come in two different types
What are the three (that i care about) Classification of mutations?
1.) Effect on structure
2.) Effect on protein sequence
In the classification that explains an effect on structure. What types of mutations are there? 6 (3+3)
In the classification that explains an effect on protein sequence. What types of mutations are there? 4
Missense (Conserve and Non-Conserve)
In the classification that explains nomenclature. What types of mutations are there? 3
Nucleotide Substitution (76A>T)
Amino acid Substitution (E6V)
Amino acid deletion (/\F508)
How does DNA sequencing work? 4
1.)Take DNA and Amplify the sample sequence using PCR (then denature it)
2.)Add Bases, DNA polymerase and fluorescently labelled di-deoxyribose nucleotides
3.) Gel electrophoresis
4.) Analyse using a computer and laser
How does DNA finger printing work (in comparision to sequencing). Basically what are the steps? 3
It takes VNTRs (variable number tendem repeats) STR (short tandem repeats) amplifies them and then gel electrophoresis.
What is reverse transcriptase PCR
Type of PCR
Uses mRNA instead of DNA and Reverse transcriptase enzymeto convert it into cDNA
How do microarrays work?
Extract either DNA or mRNA from a cell (you tend to go for one normal and one diseased cell). If mRNA you need to reverse transcriptase PCR it and ad a lable. The mix of labelled GM is added to the microarray where HYBRIDISATION occurs
Fluorescent in situ hybridisation ??
Take DNA - Denature it
Add fluorescent probe
What is the difference between an oncogene and a proto oncogene
A proto-oncogene is a normal gene that has many different functions in the cell, if it becomes mutated it will become a oncogene. Think of it like a pre-cursor
Oncogenes are genes which can transform a cell into a tumour cell
An extensive genetic test carried out on an unborn baby includes the DNA sequencing on an entire
chromosome. The test reveals a frameshift mutation which leads to a premature stop codon on one
of the alleles of a gene of known function. Explain in general terms what you expect to happen if the
mutant allele is recessive to the wild type allele, and what if the mutant allele was dominant over
the wild type allele
Dominant would mean the baby would have the diseased phenotype
Normal would mean the baby had a normal phenotype
Give an example of a environmental factor that causes single strand breaks
Reactive oxidisation species
Give an example of a environmental factor that causes doublestrand breaks