Chapter 7 DNA Fingerprinting & Crime Investigation

Isolating a DNA region of Interest within a chromosome Each chromosome is a continuous DNA molecule that is 1000s / millions of base pairs long -> a problem for scientists studying shorter stretches of DNA that makeup genes (regions of interest). Werner Arber, discovered molecular scissors that cut DNA. Using E. Coli bacteria, these enzymes are a part the enzymatic immune system that functions by recognizing & destroying foreign DNA (bacteria phages) and modifying its own DNA to prevent self-destruction (methylation).

These RESTRICTION ENDONUCLEASES are VERY specific in where they cut foreign DNA: a given enzyme will cut DNA only when it recognizes EXACTLY the right DNA sequence or restriction site.

Restriction Enzyme: molecular scissor that cuts DNA

Restriction endonuclease / enzyme (RE) cuts the phosphodiester backbone of a specific sequence in DNA. ** Sequences recognized by RE read the same from 5 to 3 as from 3 to 5 are known as a palindromic sequence. This specific recognition sequence of the RE is the restriction enzyme site".

Restriction Site

Each restriction enzyme has its own recognition sequence which after cutting will leave either a 5 overhang, 3 overhang, or blunt ends.

Cut sites

Large Scale Restriction Mapping

Large Scale Restriction Mapping Cutting up the genome into small fragments, separating them and putting the pieces into some kind of order or map based on the restriction digest pattern(s).

1) DNA Fragments of Interest

If you have long pieces of DNA (chromosomes) that are cut into smaller fragments using RE, how are you going to detect DNA fragments of your interest among the many DNA fragments/bands after gel electrophoresis?

Edward M. Southern devised a method known as Southern Blot to transfer and immobilize all the separated DNA fragments from the gel onto a DNAbinding nitrocellulose filter so that a complementary probe can detect specific DNA fragments of interest.

Radioactive-labelled probe (bait) hybridizes w complementary DNA fragments bound to nitrocellulose filter (chemical).

** Southern Blot
Southern Blotting = Restriction Enzyme Cuts + Gel Electhrophoresis + Labeled Probe Hybridization = Detection of Specific DNA Fragment

Western Blot separates protein into protein fragments & transferred to a permanent site.

2) Transfer fragments of DNA to nitrocellulose (Blotting)

Alkaline soln separates DNA into single stranded

Blotting = Mopping up the floor w a pile of dry towels water is absorbed as long as there are enough towels on top.

Now, DNA is attached to a piece of paper

3) Hybridize the DNA on the Nitrocellulose Paper to a Probe (labelled DNA).

Probe is radioactive or labelled with dye

i.

Hybridisation
The binding of two nucleic acid sequences thru H bonds in complementary base pairs

Ease of hybridisatn depends on 1) Sequence Homology (Similarity) 2) Temperature 3) Other components (Salt Concentratn in rxn)

ii.

**** Probes Labelled for Detection

Conditions: Right temp (start high & lower till hybridisatn occurs) Ingredients: Labelled probe w radioactivity, chemiluminescence (light) & Immobilised DNA

We can label a similar DNA sequence of interest (same gene for another organism) & use it as a probe for detecting that specific DNA sequence in other samples. When incubated at ~60C overnight, the probe will only bind to DNA fragments w high homology.

Radioactive Probe (CGACTGACTGAAA)

CGACTGACTGAAA ---AGTTCAAGGCTGACTGACTTTCAGT--- (High homology, strong binding)

CGACTGACTGAAA ---GCGGATTTACTGACTGACACGTTCA--- (Lesser homology, weak binding)

CGACTGACTGAAA ---TGTACAAGTCTGCGCCACTGACTGG--- (Low homology, poor binding)

Radioactive Probe added to single stranded DNA in the membrane

a) Agarose gel stained w ethidium bromide to visualize DNA fragments. b) Exposed X-ray film of a Southern blot prepared from the gel in (a). Only bands containing DNA sequences complementary to the probe show hybridization (*)

Why use Southern Blot & Probe? The sizes or # of fragments obtained gives you info about a specific region (gene) or sequence diagnose disease

Different Band patterns DNA w a mutation in the gene: A c/g of one base pair eliminates a restriction site for the RE EcoRI.

If the mutation is associated with an increased risk of an illness, it can be used for gene testing.

DNA of Wildtype gene:

** Summary for Southern Blots

1) Extract DNA from source. 2) Cut DNA into fragments (restriction enzymes). 3) Separate fragments using gel electrophoresis. 4) Transfer the fragments to a DNA-binding nitrocellulose filter & denature them w alkali (separate the 2 complementary strands). 5) Detect target DNA thru hybridizatn w a probe. 6) Remove unbound probes & detect signal

DNA Fingerprinting
Detect specific patterns of diff-size DNA fragments (polymorphisms) known to be highly variable bet individuals AND inherited from both parents in a Mendelian pattern.* Identical twins do not have same fingerprints Regions where mutations have inherited & variation is highly individualistic can generate diff sized DNA fragments after cutting w RE / Amplification by PCR.

Steps in DNA FingerPrinting 1) Extract DNA. 2) Cut DNA using restriction enzymes in conserved exons. 3) Separate DNA fragments by size on a gel. 4) **Transfer the DNA to nitrocellulose (Southern blot). 5) **Hybridize using a specific radioactive VNTR fragment as probe. **Compared to RFLP (steps 1-3 only): Gives you a smaller no of bands easier to find polymorphism (patterns of diff sizes).

Applications
1) Detecting Inherited Mutations > 90 % of our genome does not code for protein. These non-coding regions (introns) help to buffer for mutation. In the event of random mutation, there is >90 % chance that it occurs in a non-coding region so that it will not affect an encoded protein that can potentially be harmful to an individual. These intron regions become sites where mutations accumulate and passed on from one generation to another. Since DNA fingerprint is inherited, it can be used to establish a family relationship (Paternity Testing).

2) Catching Criminals Since DNA fingerprint is highly specific to each individual, it is used to match an identity w the murderer.

Mutations
Due to random mutations in each of our ancestors genomes (egg & sperm), each of us will inherit differences in DNA sequence

1) A diff # of Tandem Repeats (e.g. VNTR, STR) (repeated DNA next to each other). 2) A diff # of Restriction Enzyme Cut Sites (e.g. RFLP) Examples 1 person has 3 Tandem Repeats & another person has 10 Tandem Repeats in a specific intron. 1 person has 25 GAATTC (e.g. EcoRI restriction enzyme cut site) while another has 40 GAATTC sites. The locatn of these sites from 1 another is random & diff for each individuals genome. http://www.dnai.org/d/index.html

2 Major forms of Mutations frequently accumulated & inherited 1) Single Nucleotide Polymorphisms (SNPs) 2) Tandem Repeats: Variable Number of Tandem Repeats (VNTRs) & Short Tandem Repeats (STRs). Non coding region (intergenic) Regulatory Region (Promoter) Coding region (exon 1) Gene X Intron 1 Exon 2 Intron 2 Exon 3 Intron 3

SNPs 5 -GAATTC- 3 5 -GATTTC- 3 5 -GACTTC- 3 5- GAGTTC- 3

R1

R2

R3

R4

Tandem Repeats (R) R = 5 GATCTTTAAACCCATC- 3 (VNTR) R = 5 CATG-3 (STR)

Variations in DNA lengths usually occur in the introns (Variable Number Tandem Repeats :VNTRs)

INTRON

Only the exons get transcribed to mRNA the introns get looped out

EXON

Sequences of base pairs in repeats the no of which is inherited from parents (tandem repeats) R3 R2 R1
INTRON

R4

Cut the Introns by using Restriction Sites on the conserved (coding) exons

RFLP (Restriction Fragment - Length Polymorphism) When DNA is cut w a no of restriction enzymes => DNA fragments which vary in size (polymorphism) from individual to individual. Diff. size fragments can be separated using gel electrophoresis & visualized, or thereafter be detected using a specific probe (Southern Blotting). The banding pattern observed can serve as a DNA fingerprint of an individual (present) OR it can even be used for diagnosis of a disease (FUTURE?). **A diff. No of Tandem Repeats lead to a unique RFLP pattern.

Using RFLP to Examine Hereditary

aa

AA

Aa

AA

Using PCR to Analyse VNTRs (Nail down criminals)