Genetics Lab #8 - Forensic DNA Fingerprinting

Real world application

• Crime scene

• Paternity

• Human relatedness

• Disease-causing organisms

• Food identification

• Human remains

Deoxyribonucleic acid (DNA) structure

DNA restriction enzymes

Enzymes that destroy DNA from invading bacteriophages. Defense mechanism evolved by bacteria.

Restriction Endonucleases

Another name for DNA restriction enzyme. Makes double stranded cuts to DNA at recognition sites.

Restriction site

specific sequence where each enzyme cuts DNA. Enzymes recognize 4-6 base pair palindromic sequence

2 restriction enzymes used in lab

• EcoRI enzyme: In E. coli and contains a 5’ overhang

• Pstl enzyme: In Providencia Stuartii and contains a 3’ overhang

How do multiple fragments arise

When a specific restriction site occurs in multiple locations, a restriction enzyme (restriction endonuclease) will cut at each site producing multiple fragments 

Linear DNA fragment formula

#fragments = #restriction sites + 1

Circular DNA fragment formula

#fragments = #of restriction sites

Two types of DNA profiling

• PCR

• RFLP

Polymerase Chain Reaction (PCR)

Lab technique used to increase number of DNA copies of a specific segment from a small sample

PCR is highly specific 

• Only produces copies of desired sequence 

• Uses primers designed to be complementary and are on each side of target DNA sequence

Restriction Fragment Length Polymorphism (RFLP)

technique used to differentiate individuals by analysis of patterns derived from cleavage of DNA.

• Compares fragment sizes cut by restriction enzymes.

• Exploits variations between individuals

Restriction Fragment Length Polymorphism (RFLP) steps

• 1) Restriction enzymes cleave a region of known variability

• 2) Separating the fragments with agarose gel electrophoresis 

• 3) Determining the number of fragments and relative sizes 

What leads to differences in size or number of fragments

During RFLP mutations in nucleotide sequences within restriction sequences will lead to restriction enzymes cleaving differently.

DNA digestion reaction needs

• restriction buffer

• optimal temperature

What is within restriction buffer

• NaCl: provides correct ionic strength

• Tris-HCl: keeps pH at 8

• Mg: an enzyme cofactor

optimal temperature

37 degrees Celsius or body temp is optimal for enzymes to survive

What happens of temperature is changed

• Too Hot: enzyme will denature

• Too cold: Enzyme activity is lowered and digestion is slowed

Gel electrophoresis 

Separates DNA fragments on agarose gel and allows for fragment size determination when comapred to DNA standard 

Gel electrophoresis components

• Agarose Gel Has large pores for fragments to separate

• buffer is placed in gel to allow for current

• Samples are placed in wells with dye to track movement

• DNA ladder or strands with known band sizes must be added to 1 lane per gel

Anodes color representation on gel electrophoresis chamber

• Red is positive

• Black is negative

Which side does DNA samples move towards and why

DNA fragments have a negative phosphate backbone which will be attracted to positive end (red end) of chamber when electric current is turned on. 

Sample loading dyes use

Sample loading dyes do not stain the DNA itself but can still be used to monitor DNA movement across gel

Which fragments will move faster and farther

Smaller fragments will move faster and farther because they navigate through pores in gel better

Two different stains 

• Fast Blast DNA Stain: Dye molecules are positively charged and bind to negatively charged phosphate groups on DNA backbone. This is used to stain gel after electrophoresis has run

• SYBR or Green Glow DNA dye: Insert themselves into DNA helix and appear as a green glow when under UV light. Added to stain gel before electrophoresis.

Gel analysis

Measure the distance in mm that each DNA fragment traveled from the well and match with known base pair size. A standard curve can then be created with distance on X axis and Bp on Y axis

DNA samples used for experiment

• Crime scene

• Suspects 1-5

• DNA size standard

What is placed in each tube 

Every tube gets EcoRI/Pstl restriction enzyme mix. The DNA size standard is left as is.

What do you do with samples after adding mix

Incubate in water bath at 37 degrees Celsius for 45 minutes

Setting on electrophoresis chamber after adding DNA samples to wells

100V for 30 minutes