Flashcards in Test I Deck (37):
1
mtDNA Inheritance
In most multicellular organisms
- mtDNA is inherited from the mother (maternally inherited).
Mechanisms can involve:
- Simple dilution (Egg contains 100,000-1,000,000 mtDNA, sperm 100-1000).
- Degradation of sperm mtDNA in the fertilized egg.
- Failure of sperm mtDNA to enter the egg.
2
mtDNA and Forensics
mtDNA analysis is an appropriate method for:
- Charred remains
- Degraded specimens
- Old skeletal and fingernail samples
- Hair shafts
-Thousands of copies of mtDNA sequence in cells
-This amplification increases the chances that enough mtDNA can be obtained for forensic analysis when tissue is old or badly degraded.
-Samples either do not contain or have a marked reduction in the quality and quantity of nuclear DNA present.
3
D loop
-No info of phenotype since noncoding
-Can look at variation
-Contains hypervariable region 1 (HV1) and hypervariable region 2 (HV2) are amplified, detected, and analyzed for forensic identification
-Look at SNPs in the mtDNA hypervariable region
4
mtDNA Hypervariable Regions
-HVR1 is considered a "low resolution" region.
- HVR2 is considered a "high resolution" region.
-Getting HVR1 and HVR2 DNA tests can help determine one's haplogroup.
‘Haplogroup’ - Genetic population group of people who share a common ancestor on the direct paternal or maternal line.
5
SNPS in the mtDNA hypervariable region
-Identify remains from wars and natural disasters
-Identify the remains of the Romanov royal family assassinated during the Russian Revolution
-Determine the relationship of Ötzi, the Tyrolean Iceman, and the ancient hominid Neanderthal to modern humans.
6
How can we avoid PCR contamination?
-Designated Pipetters
-Prepare master mix (no DNA/RNA)
-UV light: maintain sterility and eliminate foreign genetic material from work stations
-The hood was a filter, prevents foreign material from entering
-Clean lab coat, gloves
-Separate rooms, one with no DNA, other with DNA. Pre and post rooms
Pre: PCR reagents
Post: PCR products
-Benches wiped with 70% ethanol to remove microbes
-Sterile disposables and reagents
7
Animal hair are classified into 3 groups
Guard hairs that form the outer coat of an animal and provide protection
Fur or wool hairs that form the inner coat of an animal and provide insulation
Tactile hairs (whiskers) that are found on the head of animals and provide sensory functions
8
SNP
-SNPs vary from person to person
-Genetic polymorphism with a population in which 2 alleles of the gene differ by a single nucleotide.
9
Nanodrop
-Shows purity and contamination
-Shows if DNA is present
-Quality and quantity
-Want a peak at 260 nm= quality is good
10
What properties of the electrophoresis buffer affect its performance during electrophoresis?
-pH
-ionic strength
-composition
11
Advantages of using a Super buffer
-DNA moves quickly through the gel
-Voltage of super buffer can can be high (200 V)
-Resolution and DNA recovery efficiency are higher than those of TAE/TBE buffers
-Can be used completely in common agarose gel electrophoresis
12
What are the two common agarose gel electrophoresis buffers used in molecular experiments?
TBE= Tris Borate EDTA
TAE= Tris Acetic acid EDTA
13
Buffer commonly used in PAGE gel electrophoresis
TBE
14
What is the purpose of the Alkaline Phosphatase?
Enzyme that catalyses the release of 5'-and 3' phosphate groups from the DNA, primers and dNTPs
15
What is the purpose of the Exonuclease1?
An enzyme that degrades single stranded DNA in a 3'-5' direction (i.e. will remove primers)
16
What alternative methods could we use to clean up a PCR reaction?
-Precipitation: removes proteins and salts
-Electrophoresis
17
What DNA sequencing services does the Waikato DNA sequencing facility offer?
Sequencing and genotyping for researchers from single stranded, double stranded, PCR derived and environmental DNA templates.
18
What is the recommended concentration of PCR products for DNA sequencing?
What concentration of sequencing primer should be supplied?
10-45 ng/mL
5 pmol/µl
2 µl of primer solution per intended reaction
19
What sequencing primers did we use to sequence the mt PCR product?
-Mito forward primer
-Mito reverse primer
20
In the electropherogram, what do internal N's in the sequence mean
N's is where the computer cannot work out what nucleotide it is
21
DNA from chewing gum
-gDNA and mtDNA from gum
Can retrieve valuable forensic information, DNA, blood group antigens and tooth inpressions
22
Proteinase K
Enzyme that degrades proteins (e.g. keratin)
-Inactivates nucleases such as DNase that could destroy the DNA
-Proteinase K is a serine protease that will hydrolyse a variety of peptide bonds
-Its active in a wide range of temps and buffers with optimal activity between 20 -60 C, pH between 7.5-12.0
23
DNA extraction from hair
-Clean hair to remove contaminants
-Digestion of hair and lysis of hair cells to liberate DNA
-Purification and/or stabilisation of liberated DNA
-Concentration and desalting of DNA extract by filtration and ethanol precipitation
Digestion of hair typically occurs in a lysis buffer which contains (pH 7.5-9), detergent, salt, chelating agent, proteinase K, reducing agent.
The main component of lysis buffer in proteinase K.
-digest hair protein keratin
-detergent that lyses cells by solubilising the proteins in the lipid membrane a disturbing the membrane interactions.
24
How can we improve our pipetting error and experimental time setup for PCR?
-Make master mix
-Liquid handling stations: automated liquid handling
-96 well plates (won't mix samples)
-Automated pipetting
25
RCF
RPM
Relative centrifugal force
Revolution per minute
26
PCR reaction
-Taq DNA polymerase
-Primers: forward and reverse
(specific for mtDNA)
-dNTPs
-Template DNA
-Mg2+ (usually in buffer)
-Sterile MQ water (make up final volume)
2X PCR Master mix solution (iTaq)
-contains Taq DNA, primers, Mg2+, buffer, dye, glycerol
-Genetic marker (forward and reverse primers)
27
PCR cycle
94 C= Denaturing
58 C= Annealing
72 C= Extending
28
SYR
-Gene: Canis familaris sex determining region Y protein
-Y chromosome
-Length 131 bp
29
CHR.X
Gene: Androgen receptor (AG) gene.
-X chromosome
-185 bp
30
STR
Single Tandem Repeats/microsatellite
-Single nucleotide repeats are 2 to 10 nucleotide sequences that are repeated in the genome and are used as genetic markers.
-No. of repeats vary between individuals so can be used as a identification
-Canines are diploid so have two sets of an STR, one from each parent
-Can be selected, amplified by PCR specific primers
-PCR products run of a PAGE gel, separates DNA based of size creating a banding formation due to diff number of repeats
-The resultant banding patterns can be compared
31
PAGE
Polyacrylamide gel electrophoresis
-High sensitivity of separation than agarose gel electrophoresis.
-Separate fragments differing 1 base pair in length
-Mainly due to uniform pore size; controlled by the concentration of acrylamide and bis-acrylamide
32
What are PEZ2, PEZ15, VWF.X and why are we using them?
-Genetic markers for canines
-Used to distinguish variation between the different dog by looking at if they contain a particular SNP.
33
Glutamate to ⍺-keto glutarate
NH4+ ⍺-keto glutarate Glutamate
NADH NAD+ Reverse Reaction
NADPH NADP+ Forward Reaction
Ammonia if too much is produced is toxic so in reverse reaction of GHD is excreted at NH4+ in the urine
34
GTP
Allosteric inhibitor
-binds to a different place than substrate and acts as an inhibitor
35
GHD
Glutamate dehydrogenase
-located in mitochondria
-catalyses the reversible NAD(P)+ of glutamate into ⍺-keto glutarate + ammonia
-Uses both NAD+ and NADP+
-Hexamer
-Allosterically regulated by the cell's energy state
ATP is high, glutamate to ⍺-keto glutarate is low
ATP is low, glutamate to ⍺-keto glutarate is high
Cofactor= NAD+, NADP+
Substrate= NH4+ and ⍺-keto glutarate
36
GHD interacting with Protein
Most proteins have pockets, can be hydrophilic/phobic
-GHD is hydrophilic, goes into the pocket.
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