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Chapter 13. DNA: The Indispensable Forensic Science Tool. Objectives:. Describe and demonstrate DNA collection, preservation, and testing, using gel electrophoresis. Use Product Rule to quantify and interpret results. Father of DNA fingerprinting. Alec Jeffreys 1985

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Chapter 13

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Chapter 13

Chapter 13

DNA: The Indispensable Forensic Science Tool


Objectives

Objectives:

  • Describe and demonstrate DNA collection, preservation, and testing, using gel electrophoresis.

  • Use Product Rule to quantify and interpret results.


Father of dna fingerprinting

Father of DNA fingerprinting

  • Alec Jeffreys

  • 1985

  • Responsible for the revelation of DNA fingerprinting


Dna fingerprinting

DNA fingerprinting

  • Isolating and reading DNA markers (portions of the DNA structure of certain genes that are unique to each individual).


What is a gene

What is a gene?

  • Fundamental unit of heredity.

  • Instructs body cells to make proteins.

  • The proteins are responsible for determining specific traits.


What is dna

What is DNA?

  • Polymer that carries the body’s genetic information.

  • Double helix.

  • Watson & Crick – built ball & stick model of DNA.


What is a polymer

What is a polymer?

  • Large molecule that links together a series of repeating units.

  • In DNA, the repeating units are called nucleotides.


What are nucleotides

What are nucleotides?

  • Building blocks of DNA molecule.

  • Composed of

    • Sugar (backbone)

    • Phosphate group (backbone)

    • Nitrogen base.


4 nitrogen bases in dna

4 Nitrogen Bases in DNA

  • Adenine + Thymine = complementary bases.

  • Cytosine + Guanine = complementary bases

  • Three nitrogen bases are called a codon.

  • Each codon codes for a specific amino acid.

  • Groups of amino acids produce a protein.


Dna to protein

DNA to Protein

  • The specific proteins produced by a cell are directly related to the sequence of nucleotides in the DNA of the cell.

  • The individuality of an organism is determined by the organism’s DNA nucleotide sequence.

  • Sickle cell anemia (example)- abnormal hemoglobin


Human genome

Human Genome

  • Total DNA content found within the nucleus of a cell.


Human genome project

Human Genome Project

  • Global effort designed to reveal the location of all genes in the human genome.

  • Will be useful in diagnosing & treating genetic diseases.

  • Will help reveal the role & implications of evolution.


Dna replication

DNA Replication

  • Synthesis of new DNA from existing DNA.

  • Semiconservative.


Steps of dna replication

Steps of DNA Replication

  • Helicase

    • Enzyme that holds double strands apart (forks)

  • DNA Primer

    • Attracts DNA polymerase

  • DNA Polymerase

    • Enzyme that inserts complementary base pair & proofreads the new DNA strand for errors.

  • Ligase

    • Enzyme that seals the new strand to the old strand of DNA.


Chapter 13

PCR

  • Polymerase Chain Reaction.

  • Technique for copying a particular DNA portion of interest outside a living cell.

  • Can produce many exact copies of segments of DNA so sample size is no longer a limitation of DNA physical evidence.

  • 1st step is to heat the DNA strands to cause them to separate completely.

  • Each cycle of the DNA Thermal Cycler takes approximately 2 minutes.


Recombinant dna

Recombinant DNA

  • The cutting of DNA into fragments that can later be incorporated into another DNA strand.

  • Enormous commercial implications (ex: growth hormones, insulin, etc)


Restriction enzymes

Restriction Enzymes

  • Chemicals that act as scissors to cut DNA molecules at specific locations.

  • Produces RFLPs.


Rflps

RFLPs

  • Restriction Fragment Length Polymorphism

  • Different fragment lengths of base pairs that result from cutting a DNA molecule.

  • All humans have the same type of repeats, but vary in the number.

  • Typically a core repeat = 15-35 bases.

  • Radioactive probes are used to visualize RFLPs (make them gel visible).


Tandem repeats

Tandem Repeats

  • Region of a chromosome that contains multiple copies of a core DNA sequence that are arranged in a repeating fashion.

  • Origin & significance is a mystery.

  • About 30% of human genome is largely composed of repeating segments of DNA.

  • It is thought that they may act as spacers between the coded regions of DNA.


Steps of dna typing

Steps of DNA Typing

  • Digestion with restriction enzymes.

  • Electrophoresis

  • Southern blotting

  • Hybridization with a radioactive probe

  • Process with X-ray film

  • In a three probe system of DNA typing, the typical DNA pattern will show a maximum of about 6 bands.

  • One band can indicate that the individuals are probably homozygous for the specific gene.


Gel electrophoresis

Gel Electrophoresis

  • DNA is separated by fragment size.

  • Smaller size fragments move faster than larger fragments.


Southern blotting

Southern Blotting

  • The transfer of DNA fragments onto a nylon membrane.


Hybridization

Hybridization

  • Process of joining two complementary strands of DNA to form a double stranded molecule.


Gene splicing

Gene Splicing

  • Laboratory procedure responsible for making possible the development of bacteria that can synthesize insulin.


Advantages of working with short dna fragments

Advantages of Working With Short DNA Fragments

  • They are more stable & less likely to break apart.

  • They are less subject to degradation due to adverse environmental conditions.

  • Quantity can be greatly amplified by PCR technology.


Chapter 13

STR

  • Short Tandem Repeats

  • Region of a DNA molecule that contains short segments consisting of 3 -7 repeating base pairs.

  • Most successful & widely used DNA profiling process.

  • Commercial STR kits include means to detect the amelogenin gene to allow for gender determination.


Str vs rflp

STR vs RFLP

  • STR analysis has replaced RFLP DNA typing because:

    • It is less subject to sample degradation.

    • It reduces the time to obtain results from a sample.

    • It requires a smaller sample size.


Str capillary electrophoresis

STR Capillary Electrophoresis

  • Evolved from flat gel electrophoresis.

  • Automates sampling and data collection.

  • Decreases analysis time.


Multiplexing

Multiplexing

  • The concept of simultaneously extracting, amplifying, and detecting a combination of STRs.

  • Y-STR markers are useful in cases where multiple males are involved in a sexual assault on a victim. (expect 3 peaks).


Mtdna

mtDNA

  • Mitochondrial DNA.

  • HV1 & HV2 are regions of mtDNA.

  • Few forensic labs do mtDNA analysis because

    • Analysis procedure is very rigorous.

    • Costs much more than nuclear DNA profiling.

    • Study takes time.

    • Discriminating power of mtDNA is less than that of STR analysis.


Dna profiling technology

DNA-profiling technology

  • Has been used to

    • Settle matters of questioned paternity/maternity.

    • Match suspect(s) to biological evidence found at a crime scene.

    • Decide immigration cases based on family relationships.


Codis

CODIS

  • Shared databases of DNA typing information from convicted felons and crime scene evidence.


Luminol

Luminol

  • Detects traces of blood without compromising potential DNA typing.


Buccal cells

Buccal Cells

  • Cells obtained from the mouth and inside of the cheek.


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