Lecture 15 Flashcards
Methods used to investigate at a protein level
- > at the structural level
- > at the functional level
Methods used to investigate at the nucleotide level (DNA level)
DNA sequencing
PCR plus sequencing
PCR plus restriction analysis
Methods used to investigate at the gene level (DNA level)
DNA sequencing
PCR technology (e.g. RT-PCR)
Southern hybridisation
Microarrays
DNA profiling
Methods used to investiage at the chromosome level
Karyotyping
Fluorescent in situ hybridisation (FISH)
Chromosome painting
Methods used to investigate at the genome level
Genome sequencing and analysis
Whole chromosome sequencing
Microarrays
Exome sequencing (looks at exons)
Microbiomes (genomes of microbs e.g. skin microbome)
PCR - the process
- Heat to 95 degrees C, this denatures DNA (breaks hydrogen bonds), seperating the two DNA strands
- Cool to 55 degrees C, so primers can anneal
- Heat to 72 degrees C, so taq DNA polymerase can catalyse the joining of dNTPs (reforming the original DNA molecule)
PCR - showing the primers - INCLUDED IN THE AMPLIFIED PRODUCT!
PCR - primers (more detail about them…)
- Forward primer and reverse primer
- Final PCR product contains the sequence of the forward and reverse primers
What do you need for PCR to occur? What is PCR aiming to do?
- Amplification of target DNA
- Repeated copying results in exponential increase in DNA
Need:
• Thermostable DNA polymerase (Taq) can also use proof reading polymerases e.g. Pfu
• Pair of primers (forward and reverse), uniquely defining the region to be copied PRIMERS ARE REALLY IMPORTANT - THEY WILL AFFECT THE REGION THAT IS BEING COPIED!
• Temperature cycles of denature, anneal, and polymerise
Aim of PCR
To amplify a specific DNA fragment
How do we know if the reaction has worked? (the sample looks the same from a naked eye, before and after!)
- Use positive (to make sure it is working, i.e. do you get the bands you expect e.g. put in forward and reverse primers for part of the chromosome you know is there, if don’t get this, look at review notes in folder) and negative controls (to see if there is contamination i.e. don’t put in the DNA template, or don’t put in the DNA polymerase - shouldn’t get any bands on the gel electrophoresis)
- Check for band of correct size using agarose gel electrophoresis
- Sequence the PCR product to check for errors
Why use PCR?
ALWAYS USED WITH OTHER TECHNIQUES E.G. GEL ELECTROPHORESIS, restriction enzyme analysis, DNA sequencing
• To amplify a specific DNA fragment
• To investigate single base mutations
e.g. Tay Sachs, Sickle Cell disease
• To investigate small deletions or insertions
e.g. Cystic Fibrosis
• To investigate variation, genetic relationships
e.g. DNA profiling, DNA typing
An example of where PCR has been used alongside gel electrophoresis
Use restriction enzyme (targeting the restirction site on the mutant allele)
- Use primers that give up to 578bp (428bp upstream and 150bp downstream)
- Amplify the DNA and restriction enzyme (not sure the order)
- Analysis the product using gel electrophoresis
For carriers:
One wild type allele will make the 578bp band, the mutant allele will make 428bp band and 150bp band due to the restriction site
Normal individual:
Two wild type alleles, only 578bp band
Homologous mutant:
Two mutant alleles: 428bp and 150bp band
LOOK AT GROUP WORK AND REVIEW NOTES - REALLY HELPFUL
What are positive controls and negative controls in PCR?
- Positive control: showing the process is working correctly, e.g. do the process with known parasitic DNA, shows that the priemrs have attached correctly etc
- Negative control: To show if there is any contamination occuring e.g. do it without DNA template or without the taq polymerase, shouldn’t get any bands in the gel electrophoresis, if do = contiaminated
What is PCR (RT-PCR)?
To determine is the gene is expressed = detected of mRNA (allows us to detect the presence of mRNA, so allows us to detect if we are getting gene expression)
What is the first step in RT-PCR?
Must convert mRNA into cDNA using the enzyme reverse transcriptase (as you can’t do PCR with mRNA as DNA polymerase doesn’t ‘work’ on mRNA)
Using PCR to make defined mutations
Incorporate mutations in DNA
Site-directed mutagenesis using modified primers
- Primers - selectively for this
- just want mutated strands at the end
What do I need to know?
- Explain the molecular basis of PCR
- Design appropriate primers to amplify a given DNA sequence
- Interpret PCR analyses used to identify common genetic diseases
- Outline the use of RT-PCR and the appropriate controls that should be used
Agarose gel electrophoresis (used with another technique!) - what is it based on? Aim of it?
- DNA is negatively charged and will move towards the positive anode if placed in an electric field
- DNA fragments can be separated on the basis of size (or shape)
Requirements for agorase gel electrophoresis
- Gel (use agarose gel in agorase gel electrophoresis):
A matrix that allows separation of DNA fragments - Buffer: maintains pH of DNA
Allows charge on the DNA samples across the gel - Power supply:
Generates charge difference across the gel - Stain/detection To identify the presence of the separated DNA
How does DNA fragments seperate?
Seperate according to size
- DNA samples (DNA fragments) are put into wells
How to visualise the DNA seperated by agorase gel electrophoresis?
Once finished gel electrophoresis, need to stain the gel in some way to be able to see the DNA molecules
• DNA is visualised by staining with ethidium bromide (makes the DNA fluorese)
• AGE is often used in conjunction with DNA fragmentation using restriction endonucleases etc
SDS-PAGE visualisation of this technique
- small proteins travel further (see this on the pic below)
SDS-PAGE - what two chemicals are required?
Proteins are complex shapes, with lots of bondings and interactions (secondary, tertiary and quarternary structures)
- *SDS:** to remove all bonds except covalent ones and disulphide ones
- *Beta-ME:** Reducing agent, reduces all disulphide bonds in the protein
As a result, the protein is turned back into it’s primary structure
What are dideoxynucleotides?
- Once bound, dideoxynucleotides terminate the chain elongation as no further nucleotides can bind
- This is because on the C 3’, this is because there is a ‘H’ on the 3’ rather than an ‘OH’
How does DNA sequencing/Sanger sequencing work?
- Based on the use of dideoxynucleotides to terminate chain elongation (once a dideoxynucleotide has been added, no further nucleotides can bind)
- This method requires breaking the DNA into many small pieces
- A dye is attached to the dideoxynucleotide (depending on it’s base - 4 different colours)
- Excess unlabeled nucleotides and fluroscently labelled dideoxy analogus added
- Once the dideoxynucleotide has bound, another nucleotide can’t join after this
- The process is repeated many times.
- By the time cycling is complete, it is gauranteed that virtually every single position has been targetted by the dideoxynucleotide
- Then it is put through gel electrophoresis, seperating the DNA strands according to length.
- A chromatogram can then be used to process this information
Another technique - DNA hybridisation - what is this?
DNA–DNA hybridization generally refers to a molecular biology technique that measures the degree of genetic similarity between pools of DNA sequences. It is usually used to determine the genetic distance between two organisms.
Use pH or heat to denature the DNA
DNA hybridisation - Southern plotting (using DNA probes) - Summary of this (diagram)
How does Southern blotting work?
The probe is complementary to the sequence that is wanted to be ‘found’ in the DNA e.g. sequence for a genetic
Southern blotting - characteristics of probes (about similarity)
• Probes do not have to have 100% similarity to the target sequence
(if 100% similarity = binds much more tightly than if there is less similarity. If a few sequences don’t much - it can still bind)
Southern blotting - characteristics of probes (about aligning)
• Probes do not have to completely align with the target sequence
Probe can still bind if has partial overlap with the target sequence (still enough complementary sequence for it to bind on to the DNA sequence)
Southern blotting - characteristics of probes (position on gel)
• Probes do not affect the position of the target sequence on a gel (won’t change the band that appears)
in pic - only see 4kb sequence, as this is the only sequence that the DNA probe is complemetary too, it is not complementary to any 2kb or the 3kb strand.
Only needs a small overlap (as said earlier)
The ‘lines’ in the strand bit on the left are where the restriction sites are, where restriction enzymes act.
LOOK AT GROUP WORK - MUCH MORE INFO ABOUT DNA probes here - e.g. how they are used when diagnosing a genetic disease etc…
DNA hybridisation - microarrays
(may need to look at hand notes for this, explained well here)
- In a cancer cell, genes can be upregulated (more mRNA than normal cells), down regulated (less mRNA than normal cells) or unchanged.
- This method shows which genes have been upregulated, downregulated or not changed.
- Microarrays allow 1000s of genes to be analysed simultaneously
- Microarrays consist of single stranded oligonucleotides with sequence corresponding to coding regions of the genome attached to a solid support
- – Normal cells and tumour cells:
- mRNA is converted into cDNA using reverse transcriptase
- Label tumour mRNA in one colour (e.g. green flureoscent label), label the nomral cell mRNA in a different colour (e.g. red fluorescent label)
- cRNA then put onto the microarray, they then bind to the mRNA in the wells.
- If in this cancer, gene A is down regulated, there will be more normal cDNA in this well than the cancer cDNA, therefore the well will be green
- If this cancer’s gene B is upregulated, there will be more tumour cDNA in this well comapred to normal cDNA. Therefore, more red than green, so the well will turn red.
- If gene C is unchanged in the cancer cell, there will be equal amounts of red and green dye in the well = colour will be between the two colours (equal green and red makes brown, so this well will be brown)
Therefore, microarrays allow you to monitor a cancer by seeing changes in gene expression. Can also work out drug targets for the cancer.
Why is FISH used?
Adding fluorescently labelled probes that can bind to target sequences on a chromosome
- Specific regions of the chromosome (e.g. to identify the presence of the chromosome, e.g. to identify if any of the chromosome has been deleted)
- Chromosome painting
FISH - e.g. help code it be used to identify Down Syndrome
- if probe for chromosome 21 is red - shows 3 chromosome 21’s are here
- if probe for chromosome 13 is green - shows 2 chromosome 13’s are here
Pic - shows this person has Down Syndrome (Trisomy 21)
FISH - can also be used to show if part of the chromosome is missing
E.g. The control probe for chromosome 7 is green = pic below shows two chromosome 7
The probe for the region that is known to be deleted in this syndrome shows that only one of the chromosome 7 has a set of this DNA in the chromosome, it has been deleted from the other chromosome
= this person has Elastin Williams Syndrome.
Note - DNA probes can also be specific for certain regions to see if they have been deleted
FISH - Chromosome painting - what is this?
Makes a probe for a whole chromosome
This pic below shows reciprocal translocation has occured, chromosome 7 (the green ones), bit is on another chromosome too…
The ball at the bottom, shows the chromosomes in there territory… (interphase)
Chromosome painting - normal and tumour
This is useful when looking at tumour cells
This shows that in a tumour cell, there is lots of duplications, translocations, rearrangements occurring…
DNA fingerprinting - what is this
DNA profiling (also called DNA fingerprinting) is the process of determining an individual’s DNA characteristics. DNA analysis intended to identify a species, rather than an individual, is called DNA barcoding.
DNA profiling is a forensic technique in criminal investigations, comparing criminal suspects’ profiles to DNA evidence so as to assess the likelihood of their involvement in the crime. It is also used in parentage testing, to establish immigration eligibility, and in genealogical and medical research. DNA profiling has also been used in the study of animal and plant populations in the fields of zoology, botany, and agriculture.
