14 Advanced Molecular Techniques Flashcards
State a few ethical issues with DNA sequencing.
- Could prevent illness later in life
- Although could encourage discrimination e.g. Insurance
- Who owns the DNA seq? You, NHS that paid, company that carried it out?
What is DNA hybridisation?
- Denature dsDNA by raising temp
- ssDNA identical to one strand added before allowing to cool and anneal
- Labelled with radioactive/fluorescent marker
- some ssDNA will anneal with original dsDNA and some wont
- Determine degree of hybridisation
What is PCR using allele specific primers?
If you know of a certain mutation, you can use a specific primer to check if the mutation is there (See notes for diagram)
- Add two primers - one complementary to wild type gene and one complementary to mutated
- See which one elongates
- Will tell us if mutation present or not
What is Sanger Chain Termination Method?
AKA DNA sequencing
- DNA denatured
- Primer annealed
- 4 reaction sequences set up with template strand+ primer
- Add DNA pol
- Free nucleotides - ddNTPs - added which will anneal to template strand but will not elongate as dd
- Products ran on gel
- Can then read sequence straight off
What is southern blotting? What is northern blotting?
DNA probes used to identify complementary DNA sequences after gel electrophoresis
- Digest DNA with restriction enzymes
- Seperate DNA by gel electrophoresis
- Transfer DNA fragments to nylon solid support
- Hybridise with labelled gene probe
- Detect hybridisation
Northern - uses DNA to detect RNA in a similar way to above
What is microarray (gene expression)?
- mRNA isolated from unhealthy and healthy cells from 2 pt
- Reverse transcriptase labelling - red for unhealthy cells, green for healthy
- Targets combined
- Hybridised to microarray
- If gene normally switched off is on, red will show
What is microarray (comparative genome hybridisation)
- Take cells from patient and extract DNA
- Label with two different fluorochromes
- Hybridise to micro array
- if green - deletion of chromosome
- red - duplication
What is DNA fingerprinting?
- 2% of our genome is coding, 98% noncoding
- Individual with two homologous chromosomes (mum and dad), will show different patterns of repeats at different loci
- These correspond to parents and can be recognised
What is karyotyping?
Identify, evaluate size, shape, number of chromosomes in body cells
- Preparation of metaphase cells
- Stained creating different banding patterns
- Line up so we can see specific patterns
E.g downs - 3 chrom of chrom 21
What is FISH?
Fluorescent in situ hybridisation
- Label DNA sequence of interest in chromosome with Fluorescent probe
- Let it hybridise
- Then you can see colour of probe that’s added and see whether green (control probe) or red (mutated/deleted gene)
What is chromosome painting?
- Normal chromosomes - all chromosomes each one colour
2. Translocations/tumour - different colours from different chromosomes connected together
What is Reverse Transcriptase PCR?
- Taq pol cant be used as not DNA, so we use reverse transcriptase to convert RNA to its DNA
- Then amplified using PCR
- Primer added to poly A tail
