Hello, and welcome back, folks, to another episode of STEM on the Streets! If you are new here, my name is Aiza, and this is my partner CAI (crime Ai), together, we stroll through the criminal-ridden streets of STEM!
If you haven't read the previous episode, click on the button to read it!
(✿◡‿◡)CAI: So, what’s your bet, Aiza? Drunk driving gone wrong, or something more... sinister?
Beats me. But you know what’s actually interesting?
(✿◡‿◡)CAI: Oh, do tell.
How they ID’d the body. They used DNA profiling. Care to guess which method?
(✿◡‿◡)CAI: Yeah, maybe—
Wrong. Let me enlighten you—
(✿◡‿◡)CAI: Aiza, I was literally about to answer.
Tsk, tsk, don't you know it is rude to interrupt?
(¬`‸´¬)CAI
Anyway! As I was saying—there are multiple types of DNA profiling, each for a different purpose. So they might have used RFLP (Restriction Fragment Length Polymorphism).
(✿◡‿◡)CAI: Hah! That is where you are wrong.
Hmm, really?
(✿◡‿◡)CAI: Yep, they probably and most likely used STR.
No, I don't think s-
(✿◡‿◡)CAI: Well then, you aren't thinking properly. You see, firstly STR is literally the golden standard in forensic DNA analysis. Secondly, the body was in water and that might have degraded the quality of DNA and we both know that RFLPs require high-quality DNA samples.
Oh, yeah! Now that I think about it, STRs are better, they are faster and require less DNA!
(✿◡‿◡)CAI: Exactly!
STR (Short Tandem Repeats)
There are multiple DNA profiling methods, each with different uses, such as STR DNA analysis, RFLP DNA analysis, Y-STR DNA analysis, and many more. However in situations such as John Conner's, STR is the most likely used.
Now, before we dive further into STRs, we must first understand a bit more about DNA. If you remember, DNA is made of nucleotides, and those nucleotides are made of bases known as nitrogenous bases: adenine (A), thymine (T), cytosine (C), and guanine (G). Now these bases make pairs (adenine pairs with thymine and cytosine pairs with guanine). These bases come in different sequences varying from person to person. For example:
ATGGCTTACCGAGGGTGA
(this sequence is only for one strand of the double helix structure of DNA)
Let's put that aside for a minute, there is something called non-coding DNA, which was previously known as junk DNA. Unlike coding DNA, non-coding DNA does not code amino acids for proteins and it is also the DNA used in STR DNA analysis.
Now that we know that, time for the STRs! CAI, that is your cue!
(✿◡‿◡)CAI: So an STR, a short tandem repeat, is a repeating sequence of bases and is only found in non-coding DNA. For example, here is a sequence of repeating bases:
CAG TGA TGA TGA CAG
Here TGA is being repeated three times, making it the STR.
Now everyone has a different number of STR repeats in their DNA and that is completely unique to them- except identical twins, who share identical DNA. STRs are inherited from either parent, each parent passes down one of their chromosomes to their child.
For example, someone could inherit:
5 STR repeats from their mother
3 STR repeats from their father.
Sometimes, parents happen to have the same number of STR repeats at a specific STR locus (a locus is like the address on a chromosome of a particular gene marker) so their child will inherit identical STR repeats making them homozygous at the locus.
Thanks, CAI! In STR analysis, what we do is that we analyze the differences or similarities between two or more DNA samples' STRs.
STR DNA Analysis
So this is how it works:
DNA Extraction
- First, the DNA is extracted from the biological sample, as I have mentioned previously, we can get DNA samples from saliva, blood, hair roots, and other biological materials and fluids. This extracted DNA is purified so that there aren't any other proteins or contaminants.
PCR
- Now this is a tad bit complicated. PCR stands for Polymerase Chain Reaction and is used to make multiple copies (amplify) STR regions :
Step 1- We must denature the DNA. This means we must separate the double-stranded DNA into single strands and break the hydrogen bonds between the base pairs. This is done by adding heat. So the DNA sample is heated to around 94-98ºC.
Step 2- Now we must prepare for the DNA extension and this process is called annealing. So the temperature is lowered again to 50-65℃ to allow primers (short DNA sequences that are fashioned to match the start and end of the targeted STR region. Primers act as the starting points for DNA extension or replication and bind to their complementary sequences on the single strand of DNA.
Step 3- After we have annealed our DNA it is time for extension or DNA synthesis. A thermophilic (heat-loving) enzyme, taq polymerase, is added so that it attaches itself to the primers and extends the DNA strands, adding nucleotides to make new DNA strands. For the taq polymerase to work at its optimum, we must raise the temperatures once more to about 72℃. The Taq polymerase creates identical copies.
Step 4- DNA extension is repeated 30-40 times to double the amount of DNA copies each cycle. And soon, after 30 cycles, over 1 billion copies are produced.
Electrophoresis
- After we have amplified our DNA, we place it on this gel plate for gel electrophoresis. Then current is added and because DNA has a slightly negative charge, the electric current pulls the DNA fragments through the gel plate and the DNA is attracted to the anode (the positive electrode). The fewer repeats or smaller STR fragments move faster and further while the longer fragments or greater repeats move slower and stay closer to wells (where they were originally loaded).
Radiation
- Now that we have the STR fragments separated on the gel plate, they are still invisible to the human eye so we transfer it to a nylon mesh. Then a short complimentary DNA sequence (DNA probe) which is radioactive or fluorescent, is added to the nylon mesh. The DNA probe binds to the STR sequences. It still isn't visible, so to see the results of the gel electrophoresis, we put the radioactive probe against an X-ray film which detects radiation, allowing us to see the DNA bands. If the probe is fluorescent, a special scanner is used.
Analysis
- Then finally you compare and analyze.
This is what a STR analysis result can look like
✿◡‿◡)CAI: It is important to remember that STR tests must be done on multiple different loci to find a complete match. For example, in America, to have a match between individuals, 13 loci must match. This varies from country to country. We must test multiple loci because there are chances that somebody's locus matches with multiple other individuals.
Well, that is all for today's episode! Next episode we'll be diving into the people who founded modern forensic DNA analysis!
This is Aiza Jamil signing out!
I am a forensics sleuth, what's your mystery to solve?
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