SESSION 12 ADVANCED MOLECULAR TECHNIQUES Flashcards
MUTATIONS CAN OCCUR AT TWO DIFFERENT LEVELS WHAT ARE THEY
MICRO- nucleotide level
MACRO- chromosome level
Can have mutations in between these two categories.
Give examples of micro mutations
- Insertion
- Deletion
- Substution
DIS
Give examples of Macro Mutation
- Deletion
- Duplication
- Inversion ( flipped over to the other side)
- Substitution
- Translocation
How can we detect DNA mutations?
- Dependent on the size of deletion you will use different techniques to determine if insertion, deletion, translocation.
DNA analysis- what techniques used to analyse DNA at NUCLEOTIDE LEVEL
- DNA sequencing ( Sanger technique - chain termination)
- PCR + restriction analysis
- PCR + DNA sequencing
( DNA sequencing not really done using the Sanger technique any more have different technique which is cheaper and faster)
WHAT IS PCR?
- Amplification of target DNA (including the primers)
- It’s just amplification so you have to combine it with other techniques after such as gel electrophoresis, restriction analysis or DNA sequencing
DNA Analysis- What techniques are used to analyse DNA at gene level?
- Southern Hybridisation ( identify DNA sequences using probes)
- Northern Hybridisation ( use DNA to detect RNA)
- RT - PCR ( REVERSE TRANSCRIPTASE PCR)
- Microarray
- DNA finger printing/ DNA profiling
WHAT DIFFERENT TYPES OF HYBRIDISATION ARE THERE?
- Southern hybridisation - use DNA probes to identify DNA sequences after gel electrophoresis.
- Northern hybridisation - use DNA to detect RNA
- Western hybridisation - * not DNA analysis - is detection of proteins by antibodies after protein gel electrophoresis
- North- western HYBDRIDISATION - detect DNA with PROTEINS
DNA analysis - WHAT TECHNIQUES ARE used to analyse DNA at the chromosome level?
- Karyotyping ( to see if there are chromosome abnormalities)
- FISH/ Chromosome painting
BRIEFLY DESCRIBE THE DNA sequencing technique
Sanger DNA sequencing
Chain termination
Have Deoxy building blocks - dCTP, dATP, dTTP, dGTP
Have dideoxy nucleotides- no OH on the 3’ or 2’ but need the OH on the 3’ for elongation of the chain via phosphodiester bond formation.
Can label the dideoxy with fluorescent tag
Look at the peaks to sequence DNA.
WHY IS GENOME SEQUENCING IMPORTANT?
- We can learn by comparing genome sequences - maybe learn about our own diseases
- eg the gorilla genome - Gorilla’s resistant to Malaria we are not although our genome is similar therefore there must be something in their DNA.
- Have MICROBIOMES - sequence of all the microbes we carry- important because we have more bacterial cells in eukaryotic cells on our body.
How has DNA sequencing changed?
- We no longer use the Sanger technique we have an other technique which is cheaper and quicker
- Sequencing much quicker can ~ 200 genomes a day
Briefly describe the two genome projects:
- 1000 genome project - looking at gene variations all over the world
- 10,000 genome project uk - Looking at individuals with many diseases and individuals who have neuronal disease ( neurodevelopment) and are extremely obese.
Explain what is meant by ethics in DNA sequencing
Different individuals would be interested in you genome information not just you family, spouse , doctors( prevent illness in later life) but police, insurance companies and the government .
- Would insurance companies charge more if your more susceptible to illness- therefore would it lead to discrimination.
- Who exactly owns the information.
WHAT IS MEANT BY DIRECT TO CONSUMER GENETIC TESTING?
- All these companies offering genetic testing from
- genetic testing of partners, paternity testing , you can even check if you have a certain sports gene. Companies looking at certain regions within a genome and can tell you something about your health.
Describe the process of RT-PCR
REVERSE TRANSCRIPTASE-PCR
- Analysis of DNA at GENE level
- need forward and revere primer - amplify the target region
- have mature MRNA - does not contain introns
- but need to covert to DNA because DNA polymerase does no bind - so use REVERSE TRANSCRIPTASE make CDNA
WHAT IS USEFUL ABOUT RT- PCR?
- Can look at gene expression to see if a certain gene is expressed you will have MRNA and if is not expressed no MRNA therefore no Amplification
( hence a technique to look at DNA at gene level)
What is meant by allele specific primers?
- In PCR you can use an oligonucleotide probe to detect specific alleles
- Dependent on the primer whether you will get the PCR product or not.
- You can have a specific rider which can give you or a band or not depending if the allele is present
- Can use allele specific probes in Southern hybridisation!
What is meant by allele specific probes?
- can use them in SOUTHERN hybridisation
- Normally you do not have probes to have complete sequence complementarity if less will bind less strongly.
- However , you can use specific conditions which affect the binding - less likely to bind if it has a mismatch in it .
What is MICROARRAY technology?
Analysis of DNA at the gene level
?- is the orderly arrangement of thousands of identified sequence genes printed on an impermeable solid support usually glass, silicon chips or nylon membrane.
- Microarray is pattern of lots of DNA on a glass or plastic called a GENE chip ( each well lots of molecules of DNA)
Can look at thousands of genes - GENOME wide analysis
DESCRIBE MICROARRAY result
- Creating microarray- analysis of ~10,000 GENES
- Genome wide analysis
- HAVE TO COMPARE TWO CONDITIONS eg. Normal and cancer cells
- Dependent on the colour if gene is switched on and off ( c and n cells)
- mixed colours means that just as much of the gene is found in c and n cells.
WHAT IS DNA finger printing?
AKA DNA PROFILING
- one the genome at different loci have microsatellites ~ 30 bp
Some regions repeated over and over again. - individuals have different repeats at the regions
- THEREFORE CAN CREATE UNIQUE. Patterns of DNA = principle of DNA fingerprinting.
- CAN be used for paternity testing because you should have bands from mom and dad
- because these are highly variable regions unlikely that someone outside the family will have the same bands.
Where has DNA finger printing used?
- In immigration cases to prove maternity
- In forensic science - murder investigations
- ( police you have the peaks with numbers telling you the number of repeats in highly variable region)
Describe Karyotyping
Analysis of DNA at chromosome level
- Metaphase spread
- Looking at banding patterns of DNA- can identify the chromosome number
- In Down syndrome can see the trisomy 21
- can see abnormalities in banding and chromosome number
What is FISH?
Analysis of DNA at chromosome level.
- Fluorescent in situ hybridisation
- Looking at where probes hybdrise in the cell do not isolate the target sequence
- Elastin Williams syndrome deletion of 25 genes you can see only one copy of the gene
Describe chromosome painting?
FISH /Analysis of DNA at chromosome level
- Regions specific to a chromosome -hence the colouring
Can detect deletions and translocation which you can see in tumor cells.
What are the ethics associated with genetic testing?
- If you are testing a baby for an autosomal dominant conditions
- You will find out if that baby has the condition if they do it tells you that the mother has the condition then her mother must have the condition.
- Eg. Huntington’s Disease- Autosomal Dominant disease- Late onset disease.
