Genes And Genomes

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RT-PCR-reverse transcirptase

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- to detect an RNA virus like HIV

*extract RNA
*make cDNA
*then PCR

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Taq polymerase

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-enzyme most active at 75'c
- no proof reading activity
- error rate high
- leaves an extra A at the 3'end

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A typical PCR reaction

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* first cycle 95'c 3 mins
* denature 95'c 1 min
*anneal primers 65'c 1 min
*extension 72'c 1 min
*30 cycles of the 3 above
*final cycle- extension 72'c for 10mins
*kept at 4'c until run on gel

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PCR- problems

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* samples are easily contaminated
*may not work if DNA is very degraded (ancient DNA)

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Critical variables for PCR

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- annealing temperature
- magnesium concentration
- number of cycles
- primers

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Genetics testing using PCR

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* Multiplex PCR -eg. DMD
* Allele- specific PCR- eg. sickle cell
* Nested PCR- eg. mycobacterum tuberculosis
* RT-PCR- to detect measure RNA- HIV
* real time PCR- quantitative

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PCR strengths and weaknesses

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Strenghts
-less time consuming
-can strat with just a few molecules of DNA
-
Weaknesses
- easily contaminated
-

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Applications of PCR

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- genetic screening
-diagnosis of microbial and viral infections
- hunman DNA profiling for paternity/relationship testing
- forensic analysis
- identification ( alive or dead)
- archaeology
- agricultural screening

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DNA polymerase is used in PCR-

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- needs to be heat resistant (thermo-stable)

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The length of PCR depends on

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number of repeats

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PCR
primers & polymerase

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primers-(short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations.

Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, by using single-stranded DNA as a template and DNA primers, which are required for initiation of DNA synthesis

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Restriction Enzymes

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- discovered and charectorised in the 1960s-70s
- provide a way of cutting DNA at specific sequences.
- used in cutting and rejoining DNA molecules
- also used to map DNA

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Types of restriction enzymes

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-Type 1-recognizes a specific sequence but cuts up to 1000 bases away
- Type 2- recognises a specific sequence and cuts at that sequence
- Type 3- recognises specific sequences but cut 24-26 bases away.

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Examples of restriction enzymes

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*EcoR1
* BamH1
* Hind111
* Sma1

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Restriction modification enzymes

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- is a system for protecting bacteria from incoming DNA
- r-m system= restriction enzymes + modifying enzyme
- incoming DNA might be bacteriophage DNA or transforming DNA.
- modification protects DNA from restriction enzymes.

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Sticky Ends

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- fragment of DNA produces by restiction enzymes (EcoR1) have protruding 5' overhang and said to have cohesive/ sticky ends.

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Blunt Ends

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- Other restriction enzymes produce blunt ends which cleave along the axis of symmetry
eg.
Sma1
5' CCC-------------GGG3'
3'GGG--------------CCC5'

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Use of restriction enzymes in cloning

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-DNA molecules can be joined together with DNA ligase.
- Sticky ends are more efficiently ligated than blunt ends

Manipulating ends- converting a sticky end to a blunt end
*end filling
*digesting the single stranded over-hang

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Setting up a restriction digest

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* 1 unit of enzymes completely cuts 1ug of DNA in 1 hour - temp is usually 37'c
*Typical reaction mix
-1ul enzyme
-1ug DNA
- 2ul Buffer concentration (x10)
-up to 20ul distilled water

* Buffers
- different enzymes required different buffers: high, medium or low salt( eg RE reguire Mg+2 ions)
* terminating the reaction
- the reaction can be terminated by adding EDTA
- Most RE can be inactivated but heating the mix to 60'c

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How do you analyze restriction digest

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Electrophoresis

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RFLPs-
restriction fragment length polymorphism

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- a difference in DNA sequences between individuals that cause a difference in restriction site
- can be detected by differences in the restriction fragments

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Two types of gene librarys

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*Genomic Library
- represent all DNA sequences
-prepared from genomic DNA

*cDNA
- represents all coding sequences
- prepared from total cellular or mRNA

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Why mRNA

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1. seclections of specific genes
2.enrichment of specific genes
3. both influnence library size (reduced)

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Southern Blots and Hybidrisation

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- method for detecting specific sequences in agarose gel
-uses of 'sequence specific' DNA probes to 'light-up' specific sequences
-

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Application for southern Blotting

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DNA fingerprinting

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Steps in southern blotting
1. pre-treatment

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1. pre-treatment
* alkali treatment
-DNA in the gel needs to be single-stranded so it can hybridise with the probe
- the gel is treated with alkali to denature the DNA
* Neutralization
- restore to nautral PH (tris buffer)

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Steps in southern blotting
2. setting up the blot- types of filter

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*Nitrocellulose
-can bind single stranded DNA, RNA and protein
- frigile
*Nylon
-can bind ssDNA, dsDNA, RNA and protein
- higher binding capacity
-not fragile

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Stringency- High

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only very specific sequences bind

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Stringency- low

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similar sequences bind

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to increase stringency

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increase washing temp

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Northern Blots

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- blots of RNA gel
- RNA is run on gel and blotted on filter
- filter is probed with DNA probe

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Northern Blot purpose

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-To identify mRNA species
- Quantify mRNA, to measure expression

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southern blot - probing

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*Hybridisation of the probe to the blot
- The blot is placed in solution containing the single-stranded probe and hybridisation is allowed to take place

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Gene cloning- Choosing a vector

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- high copy number
- 1 or more selectable markers for the identification and selection of bacterial cells containing the plasmids (ge antibotic resistant)
- low molecular weight to allow for insertion of foregin. small plasmids tend to have high copy numbers
- at least on and normally several, unique restriction enzymessites where foreign DNA can be inserted

-

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Plasmid pUC18- important characteristics

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- small
- has a col E1 replication origin
- high copy number
- has an ampicillin-resistant gene as a selectible marker
- lacZ selectable marker system
- several unique cloning sites which are clustered together in a region called polylinker or multiple cloning sites.

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Creation of recombinant plasmids containing foreign genes-

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- fragments of DNA encoding the gene of interest can be cloned into a plasmid by cleaving the plasmid at an appropriate RE site and ligation in the foreign DNA. both plasmid and foreign DNA must have compatible RE sticky ends or the ends must be blunt.

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DNA ligations

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- Phage T4 DNA encodes an enzyme T4 DNA ligase which seals single-stranded nicks between adjacent nucletides in dsDNA molecules. T4 DNA ligase requires ATP as a co-factor, ATP is hydrolysed to give an ADP-ligase complex and a free phosphate group, the complex binds to the nick in the dsDNA which must expose a 5' phoshphate and a 3' hydroxyl group so that the enzyme can make a new phosphodiesdter bond --> compleating the DNA strand.