Compares specific genetic markers, not whole genome.

Can be used for tiny samples or degraded samples because it makes many copies of sample.

Advantages of STR Analysis

1.1. Isolate Isolate DNA

2.2. Polymerase Chain Reaction (PCR) Polymerase Chain Reaction (PCR) Amplify (make copies) of specific loci

3.3. Electrophoresis Electrophoresis - Sort fragments

4. 4. Visualize Visualize fragments - can use various methods: Southern Blot, Stain Gel & UV Light, Fluorescent Tags

STR Analysis Procedure

STEP 1. Extract DNA from cell

• Break cell membrane – release nucleus

• Open nucleus - release DNA

protect DNA from enzymes

• Separate from other cell parts

• DNA must be precipitated

un-dissolved from solution

1. Heat DNA - strands unzip (denature)

2. Cool

3. Add - Primers - “starter pieces” of DNA bind (anneal)

- to complementary sequences at beginning of specific loci

- DNA polymerase - “replicator machine” - Nucleotides (AGCT) building blocks

4. Heat to 75° C – DNA polymerization

STEP 2. PCR Procedure

Make copies (extend primers)

Starting DNA Template

5’

5’

3’

3’

5’

5’

3’

3’

Add primers (anneal) 5’3’

3’5’

Forward primer

Reverse primer

DNA Amplification with Polymerase Chain Reaction (PCR)

Separate strands

(denature)

5’

5’3’

3’

Polymerase Chain Reaction

GCTATTCTGGGAGTCCAGAGTGGACGT

CGATAAGACCCTCAGGTCTCACCTGCA

TGG

CAC

G CG AT

TA C

In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created

In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created

PCR Copies DNA Exponentially through Multiple Thermal Cycles

Original DNA target region

Thermal cycleThermal cycleThermal cycle

Each cycle takes less than 2 minutes from start to finish.

STEP 3. Electrophoresis

• Southern Blot & Autoradiography – with radioactive probes & X-ray.

• Gel may be stained and visualized with UV light after electrophoresis is complete.

• Fluorescent tags can be added to primer before running gel.

STEP 4. Visualization of STR Alleles

AATG AATG AATG AATGGCTCATA CTAATGCCCGTA

For a specific loci there are many different alleles

Each allele varies in length due to the number of STR’s in the middle of the segment

Fluorescent dye labels

AATGAATG AATG AATG AATG

AATG AATG AATG AATG

AATG AATG AATG AATG

AATG AATG AATG AATG AATG AATG AATG AATG

AATG AATG

AATG AATG AATG

GCTCATA

GCTCATA

GCTCATA

GCTCATA

AATG AATG AATG AATGGCTCATA CTAATGCCCGTA

CTAATGCCCGTA

CTAATGCCCGTA

CTAATGCCCGTA

CTAATGCCCGTA

• Each person has 2 alleles for a locus. M/D

• The resulting pattern of an STR sequence for a single locus has either 1 or 2 bands

• Can be homozygous or heterozygous for each locus.

• 1 band = homozygous – both fragments same length

• 2 bands = heterozygous – different fragment lengths

13 repeats

12 repeats

11 repeats

10 repeats

9 repeats

12 repeats

8 repeats

7 repeats

6 repeats

5 repeats

4 repeats

3 repeats

How many loci were tested for each individual?

An autoradadiogram has several lanes containing DNA ladders.

Each band in these lanes contains a known length of DNA -

# of base pairs

Used for comparison to determine the length of unknown DNA bands in other lanes.

Determine # bp -

Use to determine # of STR repeats

To be a match - every fragment must match up.

Rape cases

Should this person be convicted of rape?

1. separate male faction (sperm) DNA from female faction (epithelial) DNA

2. compare with DNA from blood

Paternity cases can be used to determine the parents of a child, to identify a child’s remains, in

sibling analysis and inheritance right cases.

Is each child the biological offspring of both parents?

Remember, each line represents one allele.

Each allele for each child is inherited from one of his/her parents.

How many loci were tested for

each individual?

Combined DNA Index System

Links serial crimes & unsolved cases with repeat offenders

CSF1PO

D5S818

D21S11

TH01

TPOX

D13S317

D7S820

D16S539 D18S51

D8S1179

D3S1358

FGA

VWA

AMEL

AMEL

FBI’s CODIS DNA Database

13 STR’s