DNA Flashcards
The 4 nucleotides
Adenine (A)
Guanine (G)
Cytosine (C)
Thymine (T)
Number of chromosomes within the average human cell
46
Genes
Sections in the chromosomes that code for special functions in inheritance or in the development of an embryo after conception
Homozygous
Having two of the same allele at any given locus
Heterozygous
Have two different alleles at any given locus
Allele
Different forms of a particular gene at a particular locus
Phenotype
Observed characteristic expressed by the gene
Genotype
Alleles that make up a gene
Two types of variability in alleles
Sequence polymorphisms
Length polymorphisms
SNP
Single nucleotide polymorphisms (type of sequence polymorphism)
Tandem repeats
Base pair sequence repeats that occur next to each other without any intervening base pairs
Ex. ATC-ATC-ATC-ATC
VNTR
Variable number of tandem repeats- variation in the number of repeats occurs from one individual to the next at a particular locus
Population genetics
Determination of the frequencies with which particular genetic markers occur
Product Rule
Technique of multiplying probabilities together (pop gen)
First DNA typing method
Restriction fragment length polymorphism (RFLP)
RFLP
Restriction fragment length polymorphism- typing method where restriction enzymes are used to isolate small fragments of DNA called mini satellites or VNTRs
PCR
Polymerase chain reaction- technique used to increase the amount of DNA through amplification
STR
Short tandem repeats
Hypervariable
Having a large number of alleles
Southern Blot
Method of transferring DNA fragments to a nylon membrane after separation through electrophoresis
Probe hybridization
Technique used to visualize VNTRs after southern blots, uses radioactively labeled probes of short strands of DNA that are complementary to the core-repeating units of the VNTRs
Three steps of PCR
- Denaturation
- Annealing
- Extension
Thermal cycler
Apparatus used for PCR, capable of achieving and maintaining preset temperatures very precisely
Ingredients in a PCR tube
DNA sample, reaction buffer, polymerase enzyme, nucleotides
Taq
DNA polymerase enzyme commonly used in the USA
Heat stable 5’ to 3’ polymerase activity
Has 5’ to 3’ exonuclease activity
Does NOT have 3’ to 5’ exo proof reading
PCR Step 1
Denaturation- Heated to 94 degrees C. Double stranded DNA denatures, resulting in single-stranded DNA
PCR Step 2
Annealing/primer hybridization- primers are attached to each of the separated strands, done at 60 degrees
Primers
Short strands of synthetic DNA attached during the annealing step that mark the starting points for addition of new bases to complete the reproduction of each strand
PCR Step 3
Extension- 72 degrees C- under the influence of Taq polymerase, nucleotides are added to the primer one by one
Number of PCR cycles before double stranded target sequences exist
3
Direction of DNA synthesis
5’ –> 3’
Typical number of PCR cycles
28-32
Advantages of STRs
High variability in the population
Small size makes them less sensitive to DNA degradation
Many to choose from
Allelic ladders
Strands of DNA made up of all common alleles present at each STR locus, used for calibration
Amelogenin
Locus used to determine sex. Not an STR
HV1 and HV2
The two hyper variable regions in mtDNA
Approximate length of mtDNA
16.5k base pairs
Number of genes coded for in mtDNA
37
Nucelotides
Pentose + phosphoric acid group + nitrogenous base
Nucleosides
Sugar + nitrogenous base
Normal confirmation of DNA
B DNA- right handed helix, 10.5 pb per turn
Advantages of PCR
Amp can be done on samples containing very small amounts of DNA and degraded DNA
Relatively rapid and simple
Can serve as a starting point for the detection of virtually any kind of variation to be found in the genome
Multiplex mode- simultaneous amplification reactions
Small amount required allows portions of evidence samples to be set aside for repeat testing
Controls in PCR Reaction
- DNA extraction blank
- Positive amplification control
- Negative amplification control
DNA extraction blank
Detects contamination during extraction
Positive amplification control
Shows that master mix and thermal cycler functioned correctly
Negative amplification control
Detects contamination occurring at time of PCR set up
Progressivity
Avg # of nucleotides added before the enzyme dissociates from the DNA
