DNA typing review

What is the molecular structure of the DNA molecule and its subunits?

• DNA is a double helix- two complementary single strands held together by hydrogen bonds.

• It is a polymer, made up of monomer nucleotides.

• Each single "L-shaped" monomer nucleotide is comprised of a sugar, a phosphate, and a base (either A, T, C, or G)

• The sugar and phosphates make up the "backbone," the two outside "railings" of the ladder.

• The base pairs (A with T, C with G) make up the inside "steps" of the ladder.

What areas of the human genome are compared in forensic DNA profiling?

• Non-coding, tandem repeat regions of DNA

• NOT the sections called genes, which are used for making proteins and determining the traits of the organism

• Tandem repeats are small base sequences that repeat along the strand, back to back to back to back...

• The longer sequences that were originally used are called VNTR (Variable Number Tandem Repeats)

• The shorter sequences that are currently used are called STR (Short Tandem Repeats)

What is the importance of these sections of DNA being polymorphic?

many forms

Different people have different numbers of times the sequences repeat in their DNA.

Since these sections have many different forms across the human population, we can compare them to rule people into or out of a crime scene.

How is a person's DNA type at each locus on the DNA represented in writing?

separated by a comma.

The lower number is listed first.

If someone is homozygous and has two of the same repeat from each parent, it is that number, that number again. Ex. 14,14

How many sites/loci of DNA repeats are compared in a standard forensic DNA profile?

• 13 sites plus Amelogenin for sex determination.

How is the probability of a random match (RMP) of a DNA type calculated?

• For one single site, the frequency in the population of the 1st repeat number  x  the frequency in the population of the 2nd repeat number

• If it is homozygous, that is the final number for that site.

• If it is heterozygous, the answer above gets multiplied by 2.

• Then this answer for each site gets multiplied together for the complete RMP value.

Why is it better to compare more sites/loci than fewer in forensic work?

• It narrows down the group of possible people who could share the same profile by just random luck.

• It allows more people to be excluded from having left the sample if their DNA is different at a single site.

What is the molecular goal of gel electrophoresis used?

• To separate pieces of DNA by size

How does the process of gel electrophoresis work?

• Add the sample DNA to one end of the gel. Turn on the power.

• The DNA moves through the gel, smaller pieces going farther, faster toward the bottom.

• Compare where the pieces moved, against a size control lane, to see how many repeats are in each band on the gel.

How does the process of RFLP work?

• Combine the sample DNA with a restriction enzyme that cuts it up into manageable pieces.

• Run the sample DNA through gel electrophoresis (see above).

• Add a small piece of radioactive DNA, a probe, that has a base sequence complementary to the repeat section you are looking for. (Ex. To find CATCAT, use a piece of DNA that is GTAGTA)

• Where the small piece of radioactive DNA binds in the gel, you can “visualize” (=see) when your DNA pieces landed and how big they are (=how many repeats they have)

What is a restriction enzyme? How/why is it used in RFLP?

• A restriction enzyme cuts the DNA into fragments that more easy to work with and compare to other samples.

What is a DNA probe? How/why is it used in RFLP?

• A small piece of radioactive DNA that has a base sequence complementary to the repeat section you are looking for. (Ex. To find CATCAT, use a piece of DNA that is GTAGTA)

• Where it binds and emits energy allows you to see where the DNA piece with your repeat sections moved to and how many repeats are present.

How is a gel electrophoresis buffer dilution calculated?

• It is a ratio.

• A 50x stock buffer needs 1mL stock in a total volume of 50mL.

• Set up a cross-multiplication fraction set:

  • 1 / 50 = x / Total Volume of Buffer You Need to Make.

• Solve for x. This is how much stock solution you need.

• Then find out how much water you need to dilute it in. Use subtraction:

  • Total Volume of Buffer You Need to Make - Volume of Stock Solution You Use = Volume of Water

How is the amount of agarose and buffer calculated for a desired % agarose gel?

• A % solution is defined as g / 100mL

• Again, it is a ratio.

• A 0.6% agarose gel has 0.6g of agarose / 100mL buffer.

• If you need more or less than 100mL of it, again, set up a cross-multiplication fraction set:

  • 0.6g / 100mL = y / Total Volume of Gel You Need to Make

• Solve for y. This is how much agarose powder you should weigh out on the electronic balance.

What is CODIS?

• CODIS stands for COmbined DNA Index System.

• It is a federally-maintained DNA database obtained from crime scenes and convicted violent offenders. 

From where do the individual profiles in CODIS come?

• Local levels of government obtain DNA profiles, where they migrate to state and federal levels.

From where do the population frequencies used to calculate Random Match Probabilities of CODIS come?

• Studies published in scientific journals Ex. DNA profile of ~700 blood donors

How would a DNA profile be expected to compare across a biological parent and child?

• At every locus tested, a child should have one allele in common with each parent.

What is the molecular goal of a PCR reaction? How does the process of PCR work?

• The goal of PCR (Polymerase Chain Reaction) is to copy DNA sections of interest.

• In the case of forensic science, the sections are STRs (Short Tandem Repeat regions).

  • The starting DNA sample (either Known or Unknown sample) is put into a tube.

  • To the tube DNA primers, excess free nucleotides, and Taq polymerase enzyme are added.

  • The temperature of the tube is manipulated to bring the reaction through its different phases:

    • High heat to break the hydrogen bonds of the double-stranded DNA molecule to create two single strands.

    • Lower heat to allow the primers to bind to the single-stranded molecules, giving Taq polymerase a double-stranded spot to hook on to.

    • Free nucleotides base pair up against the open single strands, and Taq polymerase attaches the free nucleotides together in covalent bonds, creating a full strand of DNA complementary to the original single strand.

  • This results in two new, identical double-stranded molecules.

• This entire sequence is repeated about 20 times, creating 1,000,000 copies of the original DNA sample.

How do STR sections of DNA compare to VNTR sections? 

• Both are sequences of DNA that are repeated back to back to back...

• STRs are shorter starting sequences, and they are repeated a fewer number of total times on the chromosome.

• VNTRs are longer starting sequences, and they repeated a higher number of total times on the chromosome.

What is DNA polymerase? How/why is it used in PCR?

• The enzyme used in PCR reactions to copy DNA.

• It covalently attaches the free nucleotides of the growing strand, making the sugar-phosphate backbone.

• Unlike many enzymes, it is heat-stable and won't denature in the high heat cycle that initially breaks apart the double-stranded DNA into two single strands prior to replication.

What are PCR primers? How/why are they used in PCR? What two purposes do they serve?

• Small, fluorescently labeled pieces of DNA

• Their base sequence is complementary to sections just outside of the tandem repeat section of interest.

• They anneal (bind) to the single stranded DNA during the PCR reaction, giving the Taq polymerase a double-stranded place to "grab onto" and begin the replication.

• Based on their fluorescent color, they also indicate which chromosome the repeat section is from.