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Human Genomics- Lecture 2

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winniesmith2's version from 2017-10-10 22:02

Section 1

Question Answer
What is the PKa of sugar-phosphate backbone1-2. extremely charged. negative. extremely hydrophilic, loves water
How many H bonds between G and C3- harder to break
How many H bonds between T and A2
Why does structure formdue to charge of backbone, allows super stable structure to form (due to 2nd law of thermodynamics) in BETA form.
Transitionstay within type of nitrogenous base. more stable.cause less instability. more common. still follows 2nd law of thermodynamics
transversionswap nitrogenous base type
DNA wants to exist in what formB- Beta form
What is synonymous substitutionsilent
What is non-synonymous subst.replacement
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Section 2

Question Answer
Single nucleotide mutations (Micro)-Resulting in single nucleotide polymorphisms (SNPs) -Accounts for up to 90% of human genetic variations -Majority of SNPs do NOT directly or significantly contribute to any phenotypes. 1% of population.
Insertion/deletion of more than nucleotides (MEDIUM)-Tandem repeat polymorphisms. Normally around 10 bps long but this can alter drastically.
What are tandem repeatsTandem repeats are genomic regions consisting of variable length of sequence motifs repeating in tandem with variable copy number.-Used as genetic markers for DNA profiling (forensic, parentage testing) -Many cause genetic diseases: +Microsatellites (Short Tandem Repeats): repeat unit 1-6 bases long +Minisatellites: repeat unit 11-100 bases long .
Gross chromosomal aberration (Macro) Numerical aberration, Structural aberration. Both types of macro mutation are rare but often causing serious genetic diseases.
Numerical aberrationUsually caused by a failure of chromosome division, which results in cells with an extra chromosome or a deficiency in chromosomes. Gametes with these anomalies can result in conditions such as Down syndrome (who have 47 chromosomes instead of 46), or Turner syndrome (45 chromosomes). Common types of numerical aberrations are: triploidy, trisomy, monosomy and mosaicism
Repeated sequences (also known as repetitive elements, or repeats) are patterns of nucleic acids that occur in multiple copies throughout the genome
Terminal repeatsidentical sequences of DNA that repeat hundreds or thousands of times found at either end of retrotransposons or proviral DNA
Tandem repeatscopies which lie adjacent to each other, either directly or inverted
Interspersed repeats(aka. interspersed nuclear elements, a repeating element that is not adjacent)
Satellite DNAtypically found in centromeres and heterochromatin
Minisatelliterepeat units from about 10 to 60 base pairs, found in many places in the genome, including the centromeres
Microsatelliterepeat units of less than 10 base pairs; this includes telomeres, which typically have 6 to 8 base pair repeat units
Interspersed repeats (aka. interspersed nuclear elements)-Transposable elements -DNA transposons: +Retrotransposons +LTR-retrotransposons (HERVs) +Non LTR-retrotransposons: -SINEs (Short Interspersed Nuclear Elements (Alu)) -LINEs (Long Interspersed Nuclear Elements (LINE-1))
Insertion/Deletion (INDEL or DIPS) polymorphisms Often resulted from localised rearrangements between homologous tandem repeats.
Structural aberration Deletions, inversions, or translocation of large DNA fragments. Also duplication, insertion and recombination.
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Section 3

Question Answer
Repeated sequences (also known as repetitive elements, or repeats) are patterns of nucleic acids that occur in multiple copies throughout the genome
Terminal repeats identical sequences of DNA that repeat hundreds or thousands of times found at either end of retrotransposons or proviral DNA
Tandem repeats copies which lie adjacent to each other, either directly or inverted
Interspersed repeats aka. interspersed nuclear elements, a repeating element that is not adjacent.
Describe Tandem repeats -Satellite DNAtypically found in centromeres and heterochromatin. -Minisatelliterepeat units from about 10 to 60 base pairs, found in many places in the genome, including the centromeres. -Microsatelliterepeat units of less than 10 base pairs; this includes telomeres, which typically have 6 to 8 base pair repeat units.
Describe interspersed repeats -Transposable elements. -DNA transposons
(Describe DNA transposons)+Retrotransposons +LTR-retrotransposons (HERVs) +Non LTR-retrotransposons: SINEs (Short Interspersed Nuclear Elements (Alu)) LINEs (Long Interspersed Nuclear Elements (LINE-1))
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Section 4

Question Answer
Different types of length polymorphisms RFLPs, VNTRs, STRs, and Alu
RFLPs (Restriction Fragment Length Polymorphisms (variable bp))  Original method of SNP analysis
VNTRs(Variable number of tandem repeats, large size polymorphisms (10-100bp), required high quality DNA, rarely analysed in normal research/practice)
STRs(Short tandem repeats (2-13 bp multiple times))
Alu (SINE (50-500bp) and LINE (~ 7000bp) interspersed polymorphisms)
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Section 5

Question Answer
steps for obtaining a sample (biology)DNA extraction -> DNA Quantitation -> RFLP analysis/PCR Amplification
steps for obtaining a sample (technology/chemistry)Separation and detection of DNA size/PCR products -> Sample, Genotype, Determination
steps for obtaining a sample (Genetics)Collation of sample genotypes, HWE --> population comparisons/family comparisons/disease comparisons -> Disease association/ forensic/ population genetics
Sources of DNAAny nucleated cell (blood,semen, saliva, sweat,urine, other body fluids. Tissues, Hair roots, teeth/bones, faeces, dandruff, ear wax, fingerprints
What is the amount of DNA in blood30ug/mL
What is the amount of DNA in semen250ug/mL
What is the amount of DNA in salivia5ug/mL
What is the amount of DNA in hair750ng/per hair
What effects the quality of DNAthe age of the sample, biological content of cell
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Section 6

Question Answer
what are the 3 common steps/phases for extracting DNA from biological samples -Cellular lysis via disruption of cellular membranes. -Protein removal (protein denaturation). -DNA isolation from the remaining solution.
organic DNA extraction- method on powerpoint (pg20 lecture 2)The best quality of DNA, but laborious (method variable – H&S risk). Liquid-liquid extraction
Salting out (6M NaCl or 6M guanadine thiocyanate) (pg21)- method steps on power point -Good Quality DNA, less use of hazardous chemicals (many variations available): Detergent (SDS, Tween 20, Triton X-100)
0Solid Phase Silica Extraction/Spin Column (pg 22 DNA spin column technology enables nucleic acid purification by using solid phase silica extraction
Chelex (pg 23)Easy, short and quick method, suitable only for PCR
FTA® method (pg 24)Proprietary collection paper and extraction method
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Section 7

Question Answer
DNA Quantification: dsDNA. Yield Gel: -DNA separated on Agarose gel. -Stained with ethidium bromide or other intercalating DNA dyes (requires double stranded DNA). -Not specific to human DNA.
DNA Quantification: dsDNA: Spectrophotometry (more details pg 26)-DNA absorbs UV light at 260 nm. -More DNA = More absorbance.
Fluorometer based (Qubit) . (pg 27)-Uses Specific Fluorescent dyes to determine the concentration. -Specific kits available for double stranded and single stranded DNA. -Dyes fluorescence when bound to DNA, RNA or proteins. -Higher the fluorescence signal -> Higher DNA quantity.
What is PCR (28-34)-Most powerful tools in molecular biology! -Invented by Kary Mullis in 1983, resulting in his Nobel Prize in Chemistry. -In essence, this process acts as a “copying machine” for DNA: Molecular photocopier. -Laboratory method of DNA Replication.
PCR amplification -The Polymerase Chain Reaction (PCR) is a quick way to amplify (copying) a small segment (~3000 bp (or less)) of DNA. -Its operation relies on a DNA polymerase called Taq. -Taq is a polymerase from Thermophilis aquaticus, a bacterium (survives at high temperatures, 95oC)
PCR primersNormally 2 primers are required. One primes at the 5’ end of one strand, the other primes at the 5’ end of the complementary strand. Together these primers dictate the segment of DNA amplified.
PCR mixturePrimers, Taq, Buffers, dNTPs (A, G,C, T), target DNA etc.
PCR cycle Denature (95°C), Anneal (55-60°C) and Extend (72°C)
Affect of magnesium concentration The fidelity of the PCR depends on [Mg2+]. Magnesium ions have a variety of effects. Mg2+ acts as cofactor for Taq polymerase.
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Section 8