Lecture 6 - Intro to DNA Analysis Flashcards
Exposition
Forensic Science before we could manipulate DNA. DNA Analysis started with mainly looking at phenotypes. Once DNA Analysis started picking up, it flipped to looking at genotypes. And now it’s flipped again back to phenotype.
History
The structure of DNA was not discovered until 1953
No easy to way to look at sequence variation until mid to late
1980s
DNA was not used in forensics until really the late 1990s.
Before DNA
Blood typing. Matching blood type from sample to suspect. This is at best circumstantial.
A+ most common. AB rare. O- is universal donor. AB is universal acceptor.
A - 42%
B- 10%
AB - 4%
O - 44%
DNA
deoxyribonucleic acid, it codes for all of our proteins. double helix. Each base is connected to the sugar-phosphate backbone. Nucleotides: Adenine, Guanine, Cytosine, Thymine. Complementary nucleotide base-pairing. Bases linked by H-Bonds.
two major types of DNA
nuclear and mitochondrial
Mitochondrial DNA
More mitochondrial copies of DNA than nuclear copies. powerhouse of cell. generates ATP. mitochondria have their own genome, which encodes with mitochondrial proteins involved in that organ.
Mitochondria used to be their own organism and then were engulfed by another organism. Maternally inherited.
Different structure: circular.
~16,000 base pairs
Encoding ~37 genes
Nuclear DNA
Packed within chromosomes. In humans, 23 chromosomes
~3.2 billion base pairs!
Two types of chromosomes: autosomal and sex
Autosomal chromosomes: two copies per cell: half are maternally
inherited, other half paternally
Sex chromosomes: one pair, combination of X and/or Y
chromosomes
History of Forensic DNA Profiling
Figured out the structure of DNA in 1950’s… BUT no easy way to
determine its sequence directly
Early indirect methods at detecting sequence variation included
Restriction Fragment Length Polymorphism (RFLPs)
Restriction Fragment Length Polymorphism (RFLPs)
Restriction enzymes recognize different motifs, and sequence motifs. Oftentimes they’re palindromes, sometimes they’re not. Wherever that sequence is seen the enzyme will cut it. This creates fragments.
Because each person’s DNA sequence is going to differ throughout their genome, through random mutation, the number of restriction sites that a given person will have
is going to vary, which means that the fragments that are generated from the same restriction site is going to yield a different profile of fragments. These fragments are put in a plate and the smaller fragments will travel faster.
examples of restriction enzymes: TAC1, DPN2
Problems with RFLPs
It’s difficult to read the bands in the gel plates as fragments can bleed together or mix with gel if the gel hasn’t run well.
Back then you would need a lot of DNA (nanograms worth). Now, you just need a pinprick.
not individualizing (and often ambiguous)
More discriminating than blood types (multiple restriction enzymes
each with different population frequency
PCR
polymerase chain reaction
invented by Kerry Mullis (1986)
PCR exponentially increases DNA copies with each cycle. (1, 2, 4…)
Denature the strand. This creates two strands and then identical sequences are built on each of the strands. That cycle is repeated, usually between 20 and 35 times.
2^n copies of DNA are produced with n being the number of cycles.
PCR - 3 Stages
Denaturation is where you unwind and separate two strands of the double helix with high heat.
Primer Annealing - Short “primer” sequences bind to
the specific region of DNA that you’re looking to amplify. Heat is lowered.
Extension/Elongation: Nucleotides added
using sequence of
“template” strand. Heat is raised slightly but not enough to denature.
Why was PCR so important?
It mattered less and less, how much biological fluid, or how much of a biological evidence stain you had. Theoretically, if you had one copy of DNA, you could get as much DNA as you wanted from the PCR reaction. Happened in hours, not days.
Three Types of Signatures to Look At
-informative
-individualizing
Minisatellites
(VNTRs)
Microsatellites
(STRs)
Sequence
Variation Point
Mutation (SNPs)
Minisatellites
(VNTRs)
One of the first looked at.
variable number of tandem repeats
Microsatellites
(STRs)
short tandem repeats
blocks of repeating sequences that are found throughout the genome. They are primarily in non-coding regions. All of the forensic loci are non-coding regions.
These mutations happen when Taq polymerase, basically the enzyme that reproduces, as it’s reproducing and transcribing, it slips.
Number of repeating motifs at a particular loci is the signature
STRs are detected/analyzed via capillary electrophoresis
how it migrates, how fast it migrates through the gel is gonna be proportional to the number of repeats, which gives it a change in length
NUCLEAR DNA
Sequence
Variation Point
Mutation (SNPs)
sequence point variation, or small nucleotide polymorphisms
Informative loci spread out over different chromosomes
These are targeted by PCR.
Bio Review
Your DNA is transcribed.
Your DNA is converted to mRNA. Your mRNA goes to your ribosomes. Your ribosomes are what look at your mRNA and then add individual amino acids in a specific sequence that actually create the primary sequence. Then it gets folded and then it gets transported wherever it needs in the cell.
Genome in context of Forensics
A sequence has both exons coding and introns non-coding. Mutations happen more randomly in your genome in introns, so that is what we look at for forensics.
When you use mitochondrial DNA and why
Relatively small size, high abundance in human cells,
make it the target for many types of analysis
doesn’t have informative STRs. less genetic info
but many many copies
Use mito where you have little to no chance of recovering DNA.
Severely degraded remains (e.g., burned)
* Remains with limited tissue (e.g., hair, bones)
* Ancient remains
mass disasters, hair shafts, bones, teeth.
Why does mitochondrial DNA survive burning, etc?
More copies, higher chance of survival.
Trade-off in Mitochondrial DNA
Mito: Maternally inherited:
less individualizing, Fewer base pairs:
less information
Pros: More copies
per cell, Less degradation
over time
What to target depends on case circumstances and sample type.
