Forensic Lab Quiz 4

Calculate a genotype frequency when given a genotype at one locus and allele frequency tables. If the alleles are the same use…..

p X p

Calculate a genotype frequency when given a genotype at one locus and allele frequency tables. If the alleles are different use……

(p X p) X 2

Calculate a genotype frequency when given a genotype at multiple loci and allele frequency tables.

Step1: Calculate the genotype frequency for each given locus

Step 2: Multiply them together

Classify and describe electrophoresis as the method for separating DNA

Phosphate groups on the DNA backbone give DNA a negative charge.

In an electric field, DNA will migrate towards the positivve.

What kind of charge does DNA have?

Negative

Describe the capillary that DNA travels through during separation

Tubes of glass coated with a brown coating that allows them to be flexible

Very tiny and thin

Explain why smaller DNA molecules move faster than larger DNA molecules

Samples are separated through a viscous polymer filled capillary

Smaller molecules move faster through the gel

Describe how amplified product is prepared for separation

PCR product (amplified DNA)

Formamide: Keeps DNA in single stranded form for good separation

Size Standard: Used to determine the size of our PCR product (base pairs)

Also prepare tube of allelic ladder + formamide + size standard

Explain the purpose for adding formamide to amplified product

Keeps DNA in a single stranded form for a good separation.

Explain the purpose for adding size standard to an amplified product

Used to determine the size of our PCR product (base pairs)

Describe how size standard converts the time of peaks into base pair size

The solution is a two step measuring process:

1: Use a size standard to convert time (in data points) into size ( in base pairs)

2: Use an allelic ladder to convert size (in base pairs) into alleles (# of repeats)

Describe how the allelic ladder is used to convert size in bp (base pairs) to the number of repeats

Size standard is added to each sample before separation

• Reagent that contains amplified DNA of known sizes in base pairs

• For example, the size standard contains millions of copies of DNA that are 75 bp long, 100 bp long, 150bp long, etc.

Times of sample peaks are compared to times of size standard peaks

Given a drawing of sample peaks, estimate the size of sample peaks

Orange = size standard peaks

Green = sample peaks

Peaks tend to be spaced by 4’s in samples

Given a drawing of sample peaks, and an allelic ladder conversion chart, identify the allele of the sample peaks.

Size (base pairs) = Sample Size (base pairs) = Same Allele

How does fluorescent dye attach to DNA?

Fluorescent dye is attached to the primer

Explain how the instrument detects DNA

Fluorescent emissions detected by splitting light with prism and collecting data on CCD camera

The CCD camera converts light into electric singnal

Identify how the instrument detects DNA

At the detection window, the fluorescent dye is excited by a laser

Different dyes fluoresce in different colors

Unknown alleles are compared to the known alleles in the allelic ladder

Identify how a dye is excited

Dye is excited by a laser through the detection window

Explain why analyzing two or more loci in one amplification reaction is difficult

All alleles will be compared to the allelic ladder and you’ll get the unknown alleles all in one color and if one peak comes out at 194 but if multiple loci could have an allele at 194 then it’s difficult to tell which loci allele 194 came from.

Describe the two ways loci can be differentiated during detection

Primers can be labeled using different fluorescent dyes

Another way is to move the location of primers so all alleles for loci A come out after alleles for loci B

Describe the structure and packaging of mitochondrial DNA (mtDNA)

• Double Helix

• Ring of DNA

• Multiple copies in each mitochondria; multiple mitochondria in each cell

• Only 16,569 letters long

Identify the total length in base pairs of human mtDNA and the length of the area that varies among humans

D loop is 900 bases with a 1.7% difference

Describe why mtDNA from some samples can be amplified when STR amplification fails

mtDNA is used for old/degraded samples

Nuclear DNA: length is measure

mtDNA: sequence is examined

List the advantages of analyzing mtDNA

Can be used to help identify the source of degraded evidence if STRs cannot be amplified

Can be used to help identify human remains by comparing DNA to maternal relative

Can be used to identify the species of an unknown sample

Locate the maternal relatives in a family tree

Related through the mother

Describe the components of a PCR reaction amplifying mtDNA

• primers

• DNA polymerase

• dNTPs and ddNTPs

Describe what happens when a ddNTP is added to an elongated strand of DNA

Results in termination of product when ddNTP is added

Each ddNTP is labeled with a different color (A = green, T= red, G = yellow, C= blue)