L27- Advanced molecular techniques Flashcards
different molecular techniques must be employed when looking at
micro and macromutations
analysis of DNA can occur at
- nucleotide level
- gene level
- chromosome level
1.Analysis of DNA at nucleotide level
DNA sequencing
PCR plus restriction analysis/ DNA sequencing
Analysis of DNA at the gene level
- Southern hybridisation
- Northern hybridisation
- RT PCR
- Microarray
- DNA fingerprinting
Analysis of DNA at the chromosome level
- Karyotyping
- FISH/ Chromosome painting
DNA sequencing most famous method
sanger sequencing method
Sanger chain termination
dideoxy chain termintation
- allows us to work out the nucleotide sequence of a piece fo DNA (able to find micro-mutations)
sanger chain termination method
- Mixture of dNTPs added
- Deoxynucleotide triphosphate molecules (dNTP) which can be used in DNA replication (normal 3’ hydroxyl group which allows DNA polymerase to add another dNTP)
- Then one at a time different dideoxynucleotide triphosphate (ddNTP- e.g. dATP, dCTP, ddGTP or ddTTP), which contain a H at the 3’ position, are added
- DNA polymerase can use as a substrate, adding it to the growing DNA polymer, it cannot elongate on from it (no more subsequent phosphodiester bonds will form with new dNTPs)
- 4 separate tubes are used each with a unique ddNTP
- e.g. in one tube there will be dNTPs, DNA polymerase, labelled primer and ddCTP
- Incubate at 370C what do we see? Wherever C needs to be added in the sequence there is a chance that:
- i) A dCTP will be used and the chain will continue growing
- ii) A ddCTP will be used and stop further growth
- Because we have lots of the template, overtime we will see a mixture of new DNA molecule produced of different lengths depending on where the ddCTP is incorporated
- After incubation the products of the reaction are run out on a gel sing separate lanes for each reaction tubes
- This will separate the labelled fragments on the basis of size
- We are then able read off the sequence from the bottom of the gel to work out the nucleotide sequence in the newly synthesised strand
- The sequence can then be interpreted e.g. ATGCCTGCA
difference betweend deoxynucleotide triphosphoate (dNTP) and dideoxynucleotide triphosphate (ddNTP)
deoxynucleotide triphosphoate (dNTP) - which can be used in DNA replication (normal 3’ hydroxyl group which allows DNA polymerase to add another dNTP)
dideoxynucleotide triphosphate (ddNTP- e.g. dATP, dCTP, ddGTP or ddTTP) contain a H at the 3’ position
DNA polymerase can use as a substrate, adding it to the growing DNA polymer, it cannot elongate on from it (no more subsequent phosphodiester bonds will form with new dNTPs)
using genome sequencing we can look at
other animal genomes to learn about human disease
e. g. the fact gorillas have very similar genome, but are resistant to malaria
- could be used to develop strategies for humans
DNA sequencing ethical considerations
Who would be interested in your genome info?
- Family
- Potential spouse
- Doctors
- Government
- Police
- Schools
- Insurance company
Can the knowledge help prevent illness in later life?
Does it open up areas for discrimination?
Who owns DNA sequence?
PCR
Amplifies specific segments of DNA
What is needed for PCR?
1) Primers
2) dNTPs (free nucleotides)
3) Taq polymerase
4) DNA template to be copied
5) Buffer
6) Thermocycler
PCR Process
1) Denaturing (90-95)- separate double strands
2) Annealing (50-56)- temp lowered to enable primers to bind
3) Extension (72)- temp raised and new strands synthesised by polymerase
–> Cycle repeated- amount of DNA doubles every time
PCR uses what sort of primers
allele- specific primers- isolates point mutations
allele- specific primers
In this approach, the specific primers are designed to permit amplification by DNA polymerase only if the nucleotide at the 3’-end of the primer perfectly complements the base at the variant or wild-type sequences. After the PCR and electrophoresis, the patterns of specific PCR products permit the differentiation of the SNPs.
Southern blotting uses
uses DNA probes to identify complementary DNA sequences after gel electrophoresis
Northern blotting
uses DNA to detect RNA after gel electrophoresis
western uses
not a DNA hybridisation technique. Involves detection of proteins by antibodies after protein gel electrophoresis
why use soutehrn hydridisation
Investigate gene structure e.g. large deletions or duplications
To investigate gene expansions such as triplet repeats e.g. Huntington’s
To investigate mutations in genetics tests
To investigate variation, genetic relationships e.g. DNA fingerprinting
southern hydridiation allows
visualistion of specific DNA e.g. to see if individual has specific DNA that codes for a disease
southern hydridisation process
Take DNA sample and cleave using endonucleases à smaller pieces of DNA
DNA fragments are run on a gel electrophoresis which separates fragments based on charge and size
Take gel electrophoresis and transfer onto nitrocellulose filter (place on top and the fragments will transfer absorb onto the filter)
Take the filter and expose to radiolabelled / fluorescently labelled probe (ssDNA complementary to gene of interest)- hybridisation
Probe will anneal to gene of interest which has complementary DNA
Then expose filter to x-ray (radiolabelled)- will expose if gene A is present or not
probes in blotttingare either
Radiolabelled or fluorescently labelled
probes do not have to have
100% similarity to the target sequence
when probs have 100% similarity
binds tightly
when probes have 80% similarity
bind but less tightly
probes also do not have to
span the whole range of the target gene
probes will not affect
the position of the target sequence on a gel
Northern hydridisation
Similar process to southern blotting, however runs RNA instead of DNA on a gel electrophoresis – could use mRNA from cytoplasm
RT- PCR
RNA is converted to cDNA by reverse transcriptase (RT), and then the cDNA is amplified by PCR.
application of RT-PCR
- Detection of expressed genes
- Examination of transcript variants
- Generation of cDNA templates for cloning and sequencing
RT- PCR process
RNA consists of start codon AUG
Oligonucleotide primer (dT) primer added which binds to RNA poly A tail
RT and dNTPs added
RT binds to dT primer and synthesises cDNA by adding dNTPs in the 5’ to 3’ direction
cDNA amplified using Taq polymerase during PCR
what part of DNA does DNA fingerprinting use
Uses parts of non-coding regions to profile the DNA of different individuals.
why does DNA fingerprinting use DNA fingerprinting
due to high variability between individuals compared to genes
- like short tandem repeats (STR)
STR also refered to as
minisatellites
in three related individuals there genes will be
the same, but their STR will be different
- e..g different number of STR
How does DNA fingerprinting work
By removing STR (minisatellites) using restriction endonucleases
Amplifying them using PCR
Separating them based on their size using gel electrophoresis
How can DNA fingerprinting be used
can be used to detect murderers, show family relationships (paternal)
microarray
compares gene expression in 2 conditions
outline broad icroarray technique
- mRNA extracted from both healthy and diseased cell (10,000s of genes- genome wide)
- mRNA reverse transcribed into more stable cDNA
- Disease cells labelled red and healthy sample labelled green
- Both labelled samples then combined
- Sample then added to a microarray chip
- Labelled cDNA from sample bind to complementary DNA in the microarray
- Bound cDNA will bind tightly to its complementary DNA (hybridisation) on the chip, all unbound cDNA will be rinsed off
- Then chip put into electronic scanner, which activates fluorescent die
- Calculates the ratio of red to green DNA in each well, indicating which genes are expressed in the normal and diseased cell
microarray can be used to show
E.g. which genes are activated or repressed during disease
two types of microarray
Conditional gene expression microarray
Array comparative genome hydridisation
Conditional gene expression microarray
Compare cells (cancer vs normal cells) from the same individual
array comparative genome hydridiation
Extracts and labels DNA from cells form a normal individual and from an individual with a specific disease.Evaluates areas of the human genome for gains or losses of chromosome segments at a higher resolution than traditional karyotyping.
karyotyping
Cytogenetics, involving analysis of the entire chromosome complement through the microscope.
Evaluates:
Chromosome number
Chromosome structure
FISh
fluorescent in situ hydridisation
fluorescent in situ hydridisation
Uses fluorescent probes that binds to only those parts of the chromosome with a high degree of sequence complementarity. It is used to detect and localise the presence or absence of specific DNA sequences on chromosomes
à Fluorescent microscopy can then be used to find out where the fluorescent probe is bound to the chromosome (can be visualised)
Important tool for:
Understanding a variety of chromosomal abnormalities and other genetic mutation
FISH process
- Make a probe complementary to the know sequence
- When making the probe label it with a fluorescent marker
- Put the chromosomes on a microscope slide and denature them
- Denature the probe and add it to the microscope slide, allowing the probe to hybridise to its complementary site
- Wash off excess probe and observe the chromosome under fluorescent microscope
