DNA Fingerprinting & Forensic Analysis

How is DNA Typing Performed?

• Only one-tenth of 1% of DNA differs in each person; this variation can create a DNA profile of an individual

• In criminal cases, DNA samples are obtained from crime scene evidence and a suspect and analyzed for the presence of a set of specific DNA regions

Quick Review of Gel Electrophoresis: http://wps.prenhall.com/chet_saferstein_forensicscience_1/59/15206/3892793.cw/index.html

How is DNA Typing Performed?

• Portions of DNA contain sequences of letters that are repeated numerous times, called tandem repeats; act as filler or spacers between protein coding regions

• All humans have the same type of repeats, but there is tremendous variation in the number of repeats that each of us have

G C T G G T G C T G G C C T C

How is DNA Typing Performed?

• Two main types of forensic testing:– RFLP (Restriction Fragment Length Polymorphism)– PCR (Polymerase Chain Reaction) and STR (Short

tandem repeats)

DNA Typing

• Restriction Fragment Length Polymorphisms– Sequence of 15 to 35 bases in length and repeats

itself thousands of times– Each person will have a various number of

repeated sequences– DNA cut by restriction enzymes; each person will

have different fragments based on tandem repeats– Separate fragments by gel electrophoresis

DNA Typing

• RFLP– Requires a large amount of DNA– DNA must not be degraded– Crime scene evidence that is old or present in

small amounts is often unsuitable

PCR (Polymerase Chain Reaction)

Technique for making copies or amplifying a specific sequence of DNA

in a short period of time.

DNA Typing• PCR and STR (Short Tandem Repeats)– Most successful and widely used DNA profiling– Locations in the DNA with SHORT sequences that

repeat themselves– Each STR is normally 3-7 bases; only occurs in area

of chromosome around 400 bases in length– Fragments created with restriction enzyme and

multiplied by PCR– Fragments separated by electrophoresis

DNA Typing

• PCR and STR– Requires less DNA– Still effective if the DNA is partially degraded– Extremely sensitive to contaminated DNA from

the crime scene and within the laboratory

Recombinant DNA

How is DNA Typing Performed?

• It is not necessary to catalog every base pair in an individual’s DNA profile to find the unique differences

• DNA profiling depends on a small portion of the genome

• Inactive, non-protein coding regions contain repeated sequences of base pairs called variable number tandem repeats (VNTRs)

How is DNA Typing Performed?

• VNTRs– Found in non-coding regions of chromosomes– Repeated sequences between 1 and 100 base

pairs– Each person has some VNTRs inherited from each

parent; therefore no person has VNTRs identical to either parent

– VNTRs provides a scientific marker of identity known as a DNA fingerprint

How is DNA Typing Performed?

• VNTRs– Short repeated sequences, such as GTGTGT……..– The number of repeats in each run varies in

indivduals and are referred to as microsatellites or short tandem repeat

Chapter 8.3 Preparing a DNA Fingerprint

Specimen Collection• Investigators search a crime scene for any source of

human cells!• Precautions must be taken to avoid possible

contamination:– Wear disposable gloves and change frequently– Use disposable instruments when possible– Avoid talking, sneezing, and coughing to prevent

contamination with micro-droplets of saliva– Avoiding touching any area that might contain DNA while

handling evidence (face, nose, mouth, etc.)– Air-dry evidence thoroughly before packaging. Mold can

contaminate

Specimen Collection

Enemies of evidence• Sunlight, high temperatures, and moisture can

degrade DNA• Bacteria can contaminate samples

Extracting DNA for Analysis

• DNA can be extracted and purified – Chemically – using detergents to wash away

unwanted cellular material– Mechanically – using pressure to force DNA out of

a cell

Restriction Fragment Length Polymorphism (RFLP) Analysis

A. DNA is treated with restriction endonuclease (enzyme) to cut the DNA at specific points

B. Use gel electrophoresis to separate the pieces

B2. Gel is treated chemically to denature the DNA leaving two single strands; single stranded DNA is capable of binding to probes

C. Southern Blot Techniquei. Transfer fragments from gel to nitrocellulose or

nylon membrane; the exact arrangement and position of the cut pieces of DNA is preserved on the transfer membrane (the “fingerprint”)

ii. Membrane in incubated with a radioactive short strand of DNA called a probe; the probe contains the complementary sequence of bases

iii. The binding of the DNA fragment with its complementary probe is called hybridization; after hybridization, any probe that does not bond with the DNA is washed away

Southern Blot Analysisiv. X-ray (photo) film is placed on the nitrocellulose membrane;

because the target DNA, with the probe attached, is radioactive and emits particles, an image forms on the photographic film. This is called can autoradiograph

v. Each DNA sample produces an image as bands located in specific positions; it is then possible to compare two or more autoradiographs to see if the bands match

vi. RFLP is not used very much because it requires large amounts of DNA and can be easily contaminated

http://www.dnalc.org/ddnalc/resources/shockwave/southan.html

PCR and DNA Amplificationa. Locate the portions of DNA that can be useful for

comparison; primers are used to find these portions as they attach to the complementary DNA strand

b. Once the complementary strand is located, copying begins using the thermal cycler (Refer to Chapter 3 notes to review the heating/cooling cycle to amplify DNA)

c. PCR is sensitive to contamination and a small error in field or lab can result in the duplication of useless DNA

D. Dot Blot (or Slot Blot) Analysis

• Because the DNA produced by PCR is identical to the sample, gel electrophoresis is not needed to separate strands

i. DNA is applied to specially prepared blot strips; each dot on the strip contains a different DNA probe

ii. DNA probes in the “dots” are attached to an enzyme that can convert a colorless substrate (like DNA) into a colored one if binding occurs; if human DNA binds to its complementary probe, the dot changes color

http://upload.wikimedia.org/wikipedia/en/d/d9/DotBlotDemo.jpg