DNADNA

Chapter 13

What is DNA?What is DNA?

DNA StructureDNA Structure• DNA stands for deoxyribonucleic acid• Long molecule made up of nucleotides

• Consists of a 5- carbon sugar, phosphate group, and a nitrogenous base (adenine, guanine, cytosine, and thymine)

• Backbone of DNA is sugar and phosphate• Nitrogenous bases stick out

PhosphateSUGAR

G

PhosphateSUGAR

C

SUGAR Phosphate

A

SUGAR

T

DNADNA• Is found in all nucleated body cells

• White blood cells• Semen• Saliva• Urine• Hair• Root• Teeth• Bone• Tissue

• Is most abundant in buccal (cheek) cells

ChromosomesChromosomes

• Composed of tightly coiled DNA and proteins• Humans have 46 chromosomes• Only visible during cell division• Consists of 2 arms called chromatids held

together by the centromere

GenesGenes

• Are portions of DNA that code for specific proteins

• Is the fundamental unit of heredity• Each gene(s) codes for specific traits

• Eye color, hair color, height, etc

Historical InformationHistorical Information

James Watson and Francis CrickJames Watson and Francis Crick

• Determined that DNA is a double helix

Historical MilestonesHistorical Milestones

• 1988• FBI starts DNA casework

• 1991• First STR (short tandem repeat) paper

• 1998• FBI launches CODIS database

Base Pairing, Replication, and Base Pairing, Replication, and the Genetic Codethe Genetic Code

Complementary Base PairingComplementary Base Pairing

• Is based on Chargaff’s rules• Adenine pairs with Thymine• Cytosine pairs with Guanine

• Ex:) ATTGCGTAGGGCTA (TEMPLATE)

TAACGCATCCCGAT (COMPLEMENT)

DNA ReplicationDNA Replication

• Before a cell divides, it duplicates its DNA • Process called replication

• Process involves the DNA molecule separating into 2 strands and then producing two new complementary strands• Each strand of the DNA acts as a “template”

• Occurs until each chromosome is copied

DNA ReplicationDNA Replication

• Is carried out by enzymes that “unzip” the DNA molecule• Occurs when hydrogen bonds between the base

pairs are broken• Principle enzyme involved is DNA

polymerase• Polymerizes nucleotides to produce DNA• Also proofreads each DNA strand

RNARNA

• Is transcribed from DNA through the process of transcription• A goes with U instead of T• G goes with C

• After transcription comes translation• Pairing up of codons with anticodons on tRNA to

form an amino acid

Genetic CodeGenetic Code

• Proteins made by joining amino acids into chains called polypeptides

• There are twenty amino acids• Properties of proteins determined by the order

in which these amino acids are in• Language of mRNA instructions called

genetic code

Genetic CodeGenetic Code

• RNA has four bases- A, U, G, C• Genetic code read in threes

• Called a codon• Each three letters represents a specific amino

acid• Can be specified by more than one codon

DNA TypingDNA Typing

DNA TypingDNA Typing

• Is a method in which DNA is converted into a series of bands that ultimately distinguish each individual• Involves repeating segments of DNA numerous times

• Called tandem repeats• Act as fillers or spaces between the coding regions of DNA

• Only 1/10th of a single percent of DNA differs from one person to the next• Used to generate DNA profiles of an individual

DNA TypingDNA Typing

• Has coding and non-coding regions• 3% of the human DNA sequences code for

proteins• 97% is non-coding and is repetitive

• Same sequences repeated over and over• Are the tandem repeats

DNA TypingDNA Typing

• 3 processes utilized• RFLP

• Restriction Fragment Length Polymorphism• PCR

• Polymerase Chain Reaction• STR

• Short Tandem Repeats

RFLPRFLP• Restriction enzymes are used to cut DNA into smaller

fragments that can be separated and characterized for identification

• Steps involved• Isolate

• Separate DNA from the cell• Cut

• Using restriction enzymes to make shorter base strands• Sort

• By size using electrophoresis• Analyze

• The specific alleles for identification

RFLPRFLP• To visualize the RFLP’s, the fragments are

transferred to a nylon membrane that has been treated with radioactive probes containing a base sequence complementary to the RFLP’s being identified

• The nylon sheet is then placed against X-ray film and is exposed for several days• Bands appear where radioactive probes stuck to fragments

on the nylon sheet• Usually 2 bands will show up (one RFLP from each

chromosome)• Bands are analyzed to see similarities and/or differences

PCRPCR

• Is a technique used for making copies of a defined segment of a DNA molecule

• Can be valuable when the amount of evidence is minimal• Millions of copies of DNA can be made from a

single speck of blood• DNA taken from white blood cells, not red blood

cells

PCRPCR

• Steps involved• Heat the DNA strands

• Causes the strands to separate

• Cool the mixture and add a primer• Is a short sequence of base pairs that will add to its

complementary sequences on the DNA strand

• Add DNA polymerase and a mixture of free nucleotides to the separated strands

• Heat again to around 75 degrees Celsius for the completion

PCRPCR

• The outcome of one cycle of PCR is a doubling of the number of DNA strands

• Usually 25-30 cycles of PCR occur• Yield more than 1 million copies of the original

DNA molecule• Each cycle takes 2 minutes to complete

PCRPCR

• Advantages• Minute amounts of DNA may be used for

amplification• DNA degraded to fragments only a few

hundred base pairs in length can serve as effective templates for amplification

• Large numbers of copies of specific DNA sequences can be amplified simultaneously

PCRPCR

• Disadvantages• Contaminant DNA, such as those contaminated

with fungus or bacteria, will not amplify

ElectrophoresisElectrophoresis

• Is a technique used to separate DNA fragments

• An electrical current is moved through a gel substance causing molecules to sort by size

• The smaller, lighter molecules will move the furthest on the gel

• After developing, the fragments can be visualized for characterization

ElectrophoresisElectrophoresis

• Steps• Pipette the DNA• Load DNA into the gel wells• Run the gel• Observe and compare bands of DNA

Short Tandem RepeatsShort Tandem Repeats• AKA STR• Are locations on the chromosome that contain short

sequences of 2-5 bases that repeat themselves in the DNA molecule• Entire strand of DNA is less than 450 bases in length

• Is the most used procedure for DNA typing• Advantages

• Provides greater discrimination• Requires less time• Requires smaller sample size• DNA is less susceptible to degradation

Short Tandem RepeatsShort Tandem Repeats

• Procedure• Extract the gene TH01 from the sample

• TH01 has seven human variants with a repeating sequence of AATG

• Amplify the sample by means of PCR• Separate by electrophoresis• Examine the distance the STR migrates to

determine the number of times TH01 repeats

Short Tandem RepeatsShort Tandem Repeats

• Each person has two STR types for TH01• One inherited from each parent

• By continuing the process with additional STR’s from other genes, you can narrow down the probability of DNA belonging to only one person

Short Tandem RepeatsShort Tandem Repeats

• STR typing is visualized by peaks shown on a graph• Each represents the size of the DNA fragment

• The possible alleles are numbered for each location (loci)

Short Tandem RepeatsShort Tandem Repeats

• http://www.dnai.org/d/index.html

Y-STRY-STR

• Is another tool available to type STR’s located on the Y chromosome• Is male specific

• More than 20 different Y-STR markers have been identified

• Useful when multiple males are involved in a sexual assault

• Analysis will have only one band or peak• Is less complicated in appearance and interpretation

Outcomes to DNA TypingOutcomes to DNA Typing

• 3 outcomes• Match

• DNA profile appears the same• Lab determines the frequency

• Exclusion• The genotype comparison shows profile differences that can

only be explained by the two samples originating from different sources

• Inconclusive • The data does not support a conclusion as to whether the

profiles match

Types of DNATypes of DNA

• Nuclear• Found in the nucleus• Constitutes 23 pairs of

chromosomes inherited from both parents

• Each cell contains only one nuclei

• Mitochondrial• Found in the cytoplasm• Is inherited only from

the mother• Each cell contains

hundreds to thousands of mitochondria

• Can be found in skeletal remains

Mitochondrial DNAMitochondrial DNA

• Analysis of mDNA is more• Rigorous• Time consuming• Costly than nucleic testing of DNA

• mDNA is constructed in a circle or loop• 37 genes are involved in mitochondrial energy

generation• Is used when nuclear DNA typing is not possible

CODISCODIS

• Stands for Combined DNA Index System• Used for linking serial crimes and

unresolved cases with repeat offenders• Launched October 1998• Links all 50 states

• Locally, statewide, nationally• Requires >4 RFLP markers and/or 13 core

STR markers

Packaging Biological EvidencePackaging Biological Evidence

• Before packaging, photograph and record evidence on sketches

• Wearing disposable latex gloves while handling the evidence is required

• Clothing from victim and suspect with blood evidence must be collected

• The packaging of biological evidence in plastic or airtight containers must be avoided• Moisture can contribute to growth of DNA-destroying

bacteria and fungi

Packaging Biological EvidencePackaging Biological Evidence

• Each stained article should be packaged separately in a paper bag or in a well- ventilated box

• Dried blood is best removed using a sterile cotton swab lightly moistened with distilled water• Is air dried before being placed in a swab box, then a

paper or manilla envelope• All biological evidence should be refrigerated or

stored in a cool location until delivery to the lab• Standard/reference DNA specimens must also be

collected