Genomics- Lecture 3

winniesmith2's version from 2017-10-20 18:37

Section 1

Question Answer
What are transposable elementsSegments of DNA which have the ability to move to or be copied to other regions of the genome!
What are the two main types of transposable elements LINES and SINES
Shortened names for medium size transposable elements INDEL (insertion deletion) or DIPS (Deletion insertion polymorphism.
SINEs and Alus make up what % of total genomic DNA 6- 13%. -300bp per copy flanker by direct repeats
How were Alus namedfor the Alu1 restriction site within the element (cut by Alu1 restriction enzyme), Primate specific! - 1 million Alu insertions accounts for 5-7% of human genome.
How are SINE and Alus useful PCR able, biallelic, 3 genotypes only. Useful for genetic diversity analysis/forensic analysis. Diseases: Neurofibromatosis, Huntington’s Disease, Inherited breast cancer
PCR- Alu Analysis (TPA25). What and where is it? and how can it be used?-One of 28 Alu repeats (INDEL or DIPS - medium) within the TPA gene. -In a non-coding region of human DNA. -Highly conserved, Inserted in the last 1,000,000 years. -Dimorphic/Biallelic/Diallelic (+/+, +/-, -/- or II, ID, DD). -Used in population genetics, paternity analysis, and forensics.

Section 2

Question Answer
What is electrophoresis Technique to separate charged molecules in a medium/gel
How does electrophoresis workDNA is negatively charged, when current is applied, DNA moves from negative to positive end.
What is Agarose gel?Helps separate DNA fragments. -Made from seaweed -Low resolving power but broad range of separation (50 bp to 50 kb) -Buffers: TAE (tris acetate) has low buffering capacity (ionic strength); TBE (tris borate) and TPE (tris phosphate) have higher buffering capacity.
How can you then see the DNA?Visualization of DNA via fluorescence using specific Dyes: -Ethidium bromide (EB): EB is highly toxic! Many Safe dyes are Now available: SYBRSAFE.
What other types of gel are there?Polyacrylamide gel electrophoresis (PAGE): Sensitive for small sizes, but toxic
What are single nucleotide polymorphisms /describe themSingle Base Changes: A to T/C/G or vice versa. High frequency: estimated ~1 SNP/Kb (approx. 3 million/genome) -Mostly Bi-allelic, some are tri-allelic -SNPs has lower mutation rate -Many Millions are available for analysis!
How can SNPs be detected Variety of methods, including: Simple- PCR and PCR-RFLP, DNA sequencing. -Automated and large scale: Microarray: oligonucleotide arrays (many thousand SNPs can be analysed together!
What are SNPs useful for Functional change: cSNPs -> diseases, therefore they are useful in Complex Diseases, Drug Targets, Diagnostic Markers, Pharmacogenetics.
What are restriction endonucleases (enzymes)one of the primary tools in modern molecular biology for the manipulation and identification of DNA sequences. Utility lies in their specificity and the frequency with qhich their recognition sites occur within any given DNA sample
What are the steps in RFLPs (Restriction fragment length polymophisms)-Restriction endonuclease digestion (DNA cut into fragments) -Electrophoresis: Agarose or Polyacrylamide (separate fragments according to size) -Southern Blotting; transfer of DNA to membrane (move DNA fragments) -Hybridisation  Detection of Polymorphism (DNA complimentary nature)
What is RFLP-PCR-Reactions designed to produce products of different sizes after restriction enzyme cleavage. -PCR with specific primers to target the region of interest and then digestion with appropriate/different Restriction Enzymes. -Relatively inexpensive compared to other methods
Where does EcoRI recognise and where does it cutrecognises GAATTC and always cuts between G and A
Where does HaeIII recognise and where does it cutrecognises GGCC and always cuts between G and C.
What are the strengths/ advantages of RFLP-PCRSimple, Quick, Inexpensive, Effective.
What are the limitations of RFLP-PCR-Low sensitivity (need high concentration of sample/good quality). -Prior knowledge of DNA sequence. -Non – Automated -Multiple steps – long duration -End point (terminal) PCR -Labour intensive -Size-based discrimination only (some fragments too small) -Results are subject to interpretation

Section 3

Question Answer
What is RAPD-PCR Random Amplification of Polymorphic DNA
Describe RAPD-PCR-Amplified DNA is random (i.e. very limited or no previous knowledge of sequence) . -Variable number short primers (10 nucleotides (10-mer)) used with genomic DNA. -Electrophoretic banding pattern - semi-unique profile. -Amplification will takes place only of those regions of the genome that has the sequence complementary to the decaoligonucleotide at both ends. -No knowledge of the DNA sequence of the targeted genome – assumption primers will bind somewhere in the sequence.
Why would you use RAPD-PCRfor Rare species DNA, or in a system in which relatively few DNA sequences are compared (it is not suitable for forming a cDNA databank)
What are the limitations of RAPD-PCR- it relies on a large, intact DNA template sequence,so it has some limitations in the use of degraded DNA samples -Resolving power lower than targeted, species-specific DNA comparison methods, such as short tandem repeats.
How is RAPD-PCR analysed-Banding pattern = ‘RAPD loci’. -RAPD marker is a dominant markeri.e. presence of band is dominant & absence of band is recessive. -DNA bands of different sizes are assumed to be amplified product of different RAPD loci in the genome.

Section 4

Question Answer
What is ALFP-PCRAmplified Fragment Length Polymorphism (AFLP)-PCR. A highly sensitive method for detecting polymorphisms in DNA.
How does AFLP-PCR work -Digestion of total cellular DNA with one or more restriction enzymes and ligation of restriction half-site specific adaptors to all restriction fragments. -Selective amplification of some of these fragments with two PCR primers that have corresponding adaptor and restriction site specific sequences. -Electrophoretic separation of amplicons on a gel matrix, followed by visualisation of the band pattern.
What is Quantitative PCR (Q-PCR) or Real time PCR (RT-PCR)-Based on the detection and quantitation of a fluorescent reporter. -Monitors the fluorescence emitted during the reaction as an indicator of amplicon production at each PCR cycle (in real time) as opposed to the endpoint detection. -The first significant increase in the amount of PCR product (CT - threshold cycle) correlates to the initial amount of target template. -2-fold changes can be detected (highly sensitive). -Two main Methods(scientific supplier dependent – propiatary): SYBR® green TaqMan® probe
What are the advantages of Q-PCR/ RT-PCRNo gel electrophoresis, real time genotype calling, most specific, sensitive and reproducible. Excellent for gene expression analysis and genotyping.
What are the disadvantages of Q-PCR/RT-PCRNot good for multiplexing, more expensive (advanced chemistry)
How does RT-PCR work (more info on pg 24)-Uses fluorescence-labelled probes to determine the allelic composition of DNA samples. -Two fluorogenic probes, labelled with two distinct dyes, are used to discriminate between two different alleles. Single nucleotide differences . -TaqMan MGB probes are commonly used! -Contain a 5'-fluorescently labelled oligonucleotide conjugated to a non fluorescent quencher (nfq) and a minor groove binder (MGB) group at the 3' end.
Advantages of RT-PCR-Traditional PCR is measured at End-Point (plateau), while Real-Time PCR collects data in the exponential expansion phase. -Increase dynamic range of detection. -No-post PCR processing.
Disadvantages of RT-PCR-Due to its extremely high sensitivity, more error prone (genotype calling). -Requires expensive equipment and reagents. -Requires high technical skill & support. -DNA contamination.
Limitations of End-point PCR-Poor Precision -Low sensitivity -Short dynamic range -Low resolution -Non - Automated -Size-based discrimination only -Ethidium bromide for staining is not very quantitative: Results not numerical – semi quatitative? -Post PCR processing

Section 5

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