Lecture 9: DNA Flashcards
Prokaryotes
-Bacteria/ archaea
-Simplest structure
-Most basic life form
-No nucleus
Eukaryotes
-More complicated
-Bigger
-Organelles presents
-Animal/ plant/ fungi
Prokaryote genome
> Circular genome –> one or many occasionally linear
Small E. Coli K12 4639Kb-4405 gene
Extra DNA plasmids
Supercoiling
-Positive or negative due to
addition of extra turn in
double helix or removal of
turn
Packed into nucleoid
-Anchored with protanine
Genome is compact with little or no ‘extra DNA’
Plasmids can transfer from one bacterium to another
Confer antibiotic resistance/ability to use complex compounds as ‘food’
Eukaryote genome
> Wide range- unicellular and multicellular
Size of genome not related to complexity
Ratio of component parts vary
Gene- sequence of DNA or RNA that codes for a molecule (protein) that has a function
-Intron –> non-coding
section of the DNA
-Exon –> coding section of
the DNA
Pseudogene- gene sequence with no transcription
Genome wide repeat- repeated throughout genome
-Retrotransposon
»_space;Short Interspersed Nuclear Element (SINEs)
»_space;Long Interspersed Nuclear Element (LINEs)
»_space;Long Terminal Repeats (LTRs)
-Transposon
»_space;DNA that can move around the genome
Tandem repeat- Repeated immediately
Genomic DNA (gDNA)
> Linear chromosomes
Most have 2 copies of each chromosome- diploid but some (fungi mainly) haploid
One set of chromosomes from each parent
Eukaryotic genome
Packaging of chromosomes
> Linear chromosomes are bound to charged protein complex
-Termed histone protein
Histones form structure:
-Octamer- 2X H2A, H2B, H3
and H4
-H1- holds the structure
together
Chromosomes
> 22 autosomal pairs and XX/XY
Karyotype
Inherited in Mendelian fashion
Mitochondrial DNA (mtDNA)
> Small, circular DNA- 16.5 kb
Located in the mitochondria
High copy number per cell
Genes for mitochondrial function and tRNA/rRNA
Faster mutation rate than nuclear DNA
Low variation due to uni-parental inheritance
Useful in species identification
Eukaryotic genome
Chloroplast DNA (cpDNA)
> Nearly all code around 200 genes
Much larger than mtDNA- Pea 120kb
rRNA, tRNA, photosynthesis gene and ribosomal proteins
High copy number
cpDNA
-low variation due to uni-
parental inheritance (in
most instances)
-Useful in species
identification
Eukaryotic genome
Inheritance
> Dominant
Recessive
Genotype
Phenotype
Karyotype
DNA key terms
> Transcription: DNA copies to RNA
Translation: RNA produces proteins
Replication: copying and duplicating a DNA molecule
What is DNA?
> Deoxyribonucleic Acid (DNA)
Expressed graphically in different ways
What does DNA do?
> A way of carrying ‘information’
Genes within DNA code for the proteins that will be made by different cell types
A way of passing down ‘information’ for generations
Some viruses use RNA to perform same function
How does DNA do this?
> Stores the ‘information’
-Series of base pairs
-Triplet code creates amino
acids
-Amino acids for proteins
Protects the information
-Tightly wound in a double helix
-Packed onto chromosomes
-Encased in a protective membrane
DNA is…
> A long linear polymer of nucleic acids (also called nucleotides) made up of:
-A pentose sugar
-A nitrogenous base (the
sequence of the nucleic
acid)
-A phosphate group
Pentose sugar
> Pentose sugar
-5 carbon atoms
-labelled 1’-5’
-1 oxygen atom
-2’ carbon has missing
oxygen
»_space;De-oxy
Attached to sugar is the nitrogenous base
-Attached to the 1’ carbon
by N-glycosidic bond
-Exists two parent
compounds (groups)
Nitrogenous bases
> Purine (parent compound)
Adenine
Guanine
Pyrimidine (parent compound)
Cytosine
Uracil (RNA only)
Thymine (DNA only)
Phosphate groups
> Attached to C5 hydroxyl group
Gives the molecules a negative charge
Exists in different forms
Nomenclature depends on the number of phosphate groups
> 1 phosphate:
-Nucleoside 5’- phosphate or a 5’-nucleotide e.g. adenosine 5’-monophosphate (AMP)
2 phosphates:
-Nucleoside 5’-diphosphate or a 5’-dinucleotide e.g. adenosine 5’-diphosphate (ADP)
3 phosphates:
-Nucleoside 5’-triphosphate or a 5’trinucleotide e.g. adenosine 5’-triphosphate (ATP)
Nucleosides and nucleotides
> Unit consisting of base bonded to a sugar is a nucleotide
Unit consisting of a nucleotide joined to one or more phosphate groups is a nucleotide
RNA
> Another long linear polymer of nucleotides
Main differences between DNA and RNA:
-The sugar units are
different
-Uracil in RNA, thymine in
DNA
Single stranded DNA
> Nucleotides are linked by phosphodiester bonds
Bonds form between 5’ carbon of a 3’ carbon
DNA sequence code is always presented in 5’-3’ direction
Maintains integrity of stored genetic material
Double stranded DNA
> Two strands of nucleotides running anti-parallel
Bonds form between nitrogen bases
Hydrogen bonds
> A and T paired by 2 Hydrogen bonds
C and G paired by 3 hydrogen bonds
Hydrogen bonds in dsDNA
> Hydrogen bonds are weak
-It takes 4-21 kJmol-1 of
energy to break them
How is the dsDNA so stable?
-The large numbers of
hydrogen bonds within the
helix gives stability
-Forms a double helix
The double helix: Watson and Crick model
> Two, right-handed, helical polynucleotide chains are coiled around a common axis
The chains run in opposite directions
-Anti-parallel
The purine and pyrimidine bases are on the inside of the helix, the sugar-phosphate backbone is on the outside
The planes of the bases are nearly perpendicular to the helix axis, the planes of the sugars are nearly perpendicular to the helix axis, the planes of the sugars are nearly parallel to the helix axisrly parallel to the helix axis.
Size of DNA double helix
> Adjacent bases are separated by 0.34nm
There are 10 bases per turn of the helix
The helical structure repeats every 3.4 nm
The helix diameter is 2nm
Inside the helix
> The two chains are complementary due to the sequence of bases
The bases stack almost on top of each other giving stability due to 2 reasons:
-Hydrophobic effect
-stacking interactions called
van der Waals forces occur
between bases
DNA absorption spectra
> Stacked bases absorb less UV than unstacked bases (hydrochromism)
Absorption is maximal at 260nm
Separating the double helix
> Separating dsDNA into ssDNA is necessary for:
-the transcription into
mRNA
-the copying of the DNA
strand during replication
-the copying of the DNA
molecular during PCR
Separation occurs by disrupting hydrogen bonds between base pairs
Separation is called melting because it occurs abruptly at a certain temperature
Melting temperature (Tm) is defined as the temperature at which half of the helical structure is lost
DNA melting
> The higher the GC content, the higher the Tm
Single strands reassociate (anneal or hybridise) when Tm is lowered
Structure of DNA
> Ribose not deoxyribose, U instead of T
Usually single-stranded
-fold back on themselves
»_space;‘self-complementary’
regions form hairpin
loops which are double
helical
-Random coil
Different types of RNA
> Requires in protein synthesis
-messenger RNA (mRNA)
-transfer RNA (tRNA)
-ribosomal RNA (rRNA)
Required in other functional roles
-small nuclear RNA (SnRNA)
-micro RNA (MiRNA)
Messenger RNA (mRNA)
> Active during transcription
-complementary to DNA
sequence
-codes for protein
-Specific to gene
-varies in quantity dependent on gene
Transfer RNA (tRNA)
> Physical link between mRNA and amino acids
-linked to each amino acid
-3 base pair anti-codon
Ribosomal RNA (rRNA)
> Forms ribosome with protein
-interacts with both mRNA
and tRNA
-active during transcription
Molecular markers
> A loose term to describe a specific piece of DNA that gives a unique piece of information
There are hundreds of different molecular markers used in forensic science
They belong to distinct ‘classes’ and are detected using different methods
Analysis over time shows variation
Increasing use in STRs and DNA sequencing
Around same time as the field of forensic genetics was born
Each marker has its benefits and disadvantages
Three main markers
-gene regions
-SNPs
-STRs
Gene regions
> An entire gene or part of a gene sequence
Little variation in nuclear gene sequences as they’re coding
-Good for identifying
different genes
-example application- body
fluid identification
mtDNA gene sequences show more variation
-Good for identifying
differences between species
-examples application-
wildlife forensics
Genes of closely related individual will be similar
-can be used to track
ancestry
SNPs
> Single nucleotide Polymorphisms (SNPs)
Can occur In coding and non-coding regions
Some are highly informative others are not
Use of a SNP panel can be very powerful
SNPs occur through mutation events
Most common at position 3 in triple code
STR
> STRs are co-dominant
Inherited in a mendelian fashion
2 copies of a STR for each chromosome pair
STRs can exist as a number of different repeat units
-Dinucleotide
-Trinucleotide
-Tetranucleotide
-Pentanucleotide
-Hexanucleotide
Heterozygote if fragments are different sizes
Heterozygote observed as two peaks on Genetic Analyser
Homozygote if fragments are same size
Homozygote observed as single peak on genetic analyser
Methods of detection: PCR (polymerase chain reaction)
> Basis of all DNA detection approaches
Increases the number of copies of the molecular marker
Allows detection through fluorescence
Methods of detection: Fluorescence detection
> DNA can’t be observed directly
Detection is done by measuring fluorescence
Mechanism of fluorescence detection similar
Methods of detection: agarose gel
> Most basic approach
-detects genes (if primers
are specific)
-detects different sizes
fragments
DNA is negatively charged
DNA is run through a matrix (agarose)
DNA is separated according to its relative mobility through the matrix
Small bands travel quickly
Large bands travel slowly
Compared to a ladder
Precision is poor
The agarose gel contains an intercalating dye
The dye binds in the groves of the double helix
The dye fluoresces in UV light when bound
Methods of detection: fragment length analysis
> Same principles as agarose
Sizing accuracy is better
Can be used to detect STRs
Use a genetic analyser
Methods of detection: DNA sequencing
> Same principles as FLA
Uses a Genetic Analyser
Requires detection of single base pairs
Up to 800p reads
Detects SNPs and Gene regions
First PCR produces DNA fragment
Second (sequencing) PCR adds fluorescent ddntp
Results in multiple different length DNA strands
Each strand has different terminal coloured dye
Methods of detection: qPCR
> Combined the principles of PCR and agarose gel
Fluorescence is detected as DNA is amplified
Fluorescence is detected by using an intercalating dye
Detects Gene regions (is primers are specific)
Or molecular probe (TaqMan)
Detects Gene regions as probe is highly specific to gene
Methods of detection: melt curve PCR
> Uses the same PCR instrument
Fluorescence is detected at the end of the PCR
Detects Gene regions and SNPs
Fluorescent probes bind to all the dsDNA that has been produced
The probe fluoresces when bound
The mix is then heated
At a specific temperature the probe ‘melts’ away from the DNA
A reduction In fluorescence is observed
Used in body fluid identification
