Lecture 6 - Intro to DNA Analysis

Question 1

Exposition

Answer 1

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.

Question 2

History

Answer 2

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.

Question 3

Before DNA

Answer 3

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%

Question 4

DNA

Answer 4

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.

Question 5

two major types of DNA

Answer 5

nuclear and mitochondrial

Question 6

Mitochondrial DNA

Answer 6

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

Question 7

Nuclear DNA

Answer 7

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

Question 8

History of Forensic DNA Profiling

Answer 8

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)

Question 9

Restriction Fragment Length Polymorphism (RFLPs)

Answer 9

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

Question 10

Problems with RFLPs

Answer 10

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

Question 11

PCR

Answer 11

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.

Question 12

PCR - 3 Stages

Answer 12

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.

Question 13

Why was PCR so important?

Answer 13

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.

Question 14

Three Types of Signatures to Look At

Answer 14

-informative
-individualizing

Minisatellites
(VNTRs)

Microsatellites
(STRs)

Sequence
Variation Point
Mutation (SNPs)

Question 15

Minisatellites
(VNTRs)

Answer 15

One of the first looked at.
variable number of tandem repeats

Question 16

Microsatellites
(STRs)

Answer 16

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

Question 17

Sequence
Variation Point
Mutation (SNPs)

Answer 17

sequence point variation, or small nucleotide polymorphisms

Question 18

Informative loci spread out over different chromosomes

Answer 18

These are targeted by PCR.

Question 19

Bio Review

Answer 19

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.

Question 20

Genome in context of Forensics

Answer 20

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.

Question 21

When you use mitochondrial DNA and why

Answer 21

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.

Question 22

Why does mitochondrial DNA survive burning, etc?

Answer 22

More copies, higher chance of survival.

Question 23

Trade-off in Mitochondrial DNA

Answer 23

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.