DNA

How Do Cells Know What To Do?• Inside the nucleus of any cell is a set of

chromosomes that contain all the genetic material needed to control cell behavior and function.

• On the chromosomes are genes that are the fundamental unit for heredity.

• Genes control what proteins are made and how they work.

• Each gene is composed of a strand of DNA which makes the proteins directly.

What is DNA?• DNA is a polymer made up of thousands of

monomers called nucleotides.• A nucleotide has a sugar and phosphate (PO4

3-) backbone, and a nitrogen containing molecule called a base.

• There are 4 possible bases: Adenine (A), Cytosine (C), Guanine (G), and Thymine (T).

• The base pairs can be attached in any order, and in theory, the DNA can go on forever.

• The strand is actually a double helix.

Complimentary Base Pairing• When the DNA forms the double helix, the bases

are forced to pair. • The bases can only pair up with A-T and G-C

pairings.

Protein Synthesis• Proteins are composed of 20 amino acids.• Every three base pairs on the DNA strand will

code one amino acid.• In actuality, the DNA is used to make RNA which

then codes the protein in another part of the cell, but we can skip the middle man here.

• The order of all the base pairs on the DNA then determines the order of the linking of the amino acids on the protein which determines the function of the protein.

DNA Replication• Many proteins and enzymes help in unwinding

the DNA.• The two strands are then held apart by some

other enzymes and proteins, and are exposed to free nucleotides.

• These nucleotides are then attached to the original strand in the correct order with the help of polymerases (enzymes that make polymers)

• The result is two new strands of DNA.

How Is DNA Inherited?• During meiosis, the chromosomes break into 2

pieces, so each sex cell only has 1 strand of DNA.• Upon fertilization, the chromosome half from the

father joins the chromosome half of the mother.• In this way, the genetic make-up of the baby is

different from both but shares some characteristics.

• Each section of DNA could appear twice and be different since one piece comes from each parent.

What Can Forensics Use• It was discovered that potions of DNA have

repeating patterns.• In fact 30% of the human genome is composed of

repeating patterns• The patterns, called tandem repeats, contain no

functional use and seem to act as fillers between the coding portions of DNA.

• When a restriction enzyme is introduced, the repeats can be cut out.

• The tandem repeats are different for each individual and provide the “fingerprint.”

RFLP• Stands for Restriction Fragment Length

Polymorphisms.• The restriction enzyme acts as a scissor and cuts

out the specific repeat.• These repeats are 15-35 bases long and repeat up

to 1000 times.• The lengths of the repeating patterns are what is

different between people.• Looking at several different patterns (loci) present

in different parts of the chromosomes is how a direct identification can be made.

Gel Electrophoresis• Once the fragments are removed they need to be

separated.• They are placed on an agar gel plate and put into a

machine that has an electrical potential.• The fragments separate by length since smaller

fragments move faster.• The gel is then placed on a nylon membrane and a

radioactive DNA probe is added to bind to specific fragments.

• The radiation develops an x-ray film to provide the picture of the fingerprint.

Identification• Since genetic material comes from both parents,

there might be two different lengths for each pattern. (They could both be the same.)

• The probe can be selected to chose patterns that are only common to 1/100 of the population.

• If you choose 4 probes, you would end up with a 1:100 million combined frequency.

• RFLP is very time consuming, but can also be very powerful.

• OJ Simpson was 1 in 57 billion.

Problems With RFLP

• Time consuming.• Susceptible to damage from the elements.• Required large amounts of genetic material to

be testable.

PCR• Stands for Polymerase Chain Reaction.• DNA polymerase is an enzyme that can synthesis

specific regions of DNA.• DNA is heated to 94oC which causes the double

helix to split.• Mixture is cooled to 60oC.• Primers are added which are short DNA segments

that act as the endpoints for the region of interest.• Polymerase is added with amino acids at 72oC.• Rest of chain is filled in.

Advantages of PCR• The result of the cycle is two new strands of DNA.• Typically 28-32 cycles are done to create more

than 1 billion copies.• Each cycle is only about 2 minutes.• These can then be tested.• PCR can’t be used with RFLP because the pieces

are much shorter.• Shorter segments are less likely to break down.• The ability to reproduce DNA means that a single

cell could be enough to identify with.

STR• Stands for Short Tandem Repeats.• Shorter repeat sections of the chromosome are

identified.• There are more shorter repeats so more can be

searched for and used as markers.• Called “multiplexing” when multiple STR’s can be

looked for at one time.• CODIS is the federal database and uses 13 STRs.• PCR is suited for replicating these shorter patterns.

Separation Technique• Capillary Electrophoresis is used.• Sample is injected into a glass column coated with

the gel that has a potential across it.• The potential will cause smaller STRs to move

faster.• The material travels and goes through a detector.• The detector prints an electropherogram which

looks similar to the chromatographs we viewed earlier.

Advantages of STR• It is faster.• Can focus on loci from only the Y-chromosome to

separate out male DNA from female.• Good for rape cases or other situations of mixing

of fluids.• Can also search for amelogen gene.• Different lengths on X and Y chromosomes.• Since only males have the Y, shows two bands for

men (X and Y) and only one band from a women (X and X).

Mitochondrial DNA• All other DNA uses nuclear DNA, but mitochondria have

their own DNA.• Only comes from mother, none from father.• There are hundreds to thousands of mitochondria in each

cell and each contains several strands of DNA.• So small amounts of cells provide huge amounts of DNA.• More likely that this DNA can be found even if nuclear

DNA has been degraded.• Very expensive and tedious.• Can only tell common vs. rare. Can’t distinguish siblings.

Crime Scene Collection• DNA can be degraded and contaminated.• Biological material can be harmful so gloves must

be worn.• Always wear a different set of gloves for each

sample.• Use different forceps for picking up each sample.• Place in a ventilated storage container to prevent

moisture build-up and growth of DNA destroying bacteria.

• Obtain a standard by rubbing a cotton swab on the inside of the cheek of all suspects.