DNA: Review, Replication, & Analysis

Two types of DNA

• Nucleic DNA– Found in the nucleus of a cell– Specific to an individual

• Mitochondrial DNA (mtDNA)– Found in the mitochondria– From the maternal side– Not as specific – shows maternal side only

DNA is made of nucleotides (A, C, G, & T) that are anti-parallel

3’

3’5’

5’Coding strand

Complimentary strand

Replication/Transcription direction

DNA molecular structure & Make-up3’-OH

5’- P

3’-OH

5’- P

H-Bond

A

T

CG

T

A

GC

Transcription/ replication direction

Complimentary strand

Deoxyribose sugar

Phosphate group

nucleotides

Chromosomes1 from each parent

Sex-chromosomes

Autosomes

Nuclear DNA

3.2 billion bp Located in cell nucleus

2 copies per cell

Chromosomes

• Locus (pl. loci) specific location on the molecule (DNA) that contain:

– Genes – unit of heredity• Made of different alleles

– Homozygous = alleles are the same

– Heterozygous = alleles are different

Vocab Review• Recombinant DNA - opening up the base

pr. Of the helix and recombining it with another strand

• Restriction enzymes - chemicals that cut DNA into fragments that can later be incorporated into another DNA strand; ~150 different kinds

• Polymer – long-chained molecule (e.g. DNA)

Vocab Review

• Probe - single strand of nucleic acid, much like RNA that has been made in a way that its base sequence lines up to hybridize areas on an allele; usually labeled w/ radioactive material

• Polymerase - enzyme that is used to assemble new strands of DNA to the original/parent strand

RFLP

• Restriction Fragment Length Polymorphisms– Fragment lengths of repeating bases result from

using restriction enzymes

• 1st method used in forensic science

RFLP

RFLP Process

• Need large amount of DNA• DNA is placed w/ restriction enzyme• Cut DNA is then separated using

electrophoresis• DNA bands transferred to Nylon Membrane

(Southern blotting) • Radioactive DNA probe is added to

membrane (process called hybridization)

RFLP Process (cont.)

• X-ray film placed next to membrane for a couple of days

• X-ray DNA film fragments then measured samples along with control (comparison)

• RFLP strands used are typically thousands of bases long

PCR DNA typing technique• Polymerase Chain Reaction

• Now being used more than RFLP

• Requires only small amount of DNA

• Produces large amount of DNA

• Can be used to aid other techniques

• Uses electrophoresis

• Best on strands no longer than a couple of hundred bases long

PCR Process• Heat DNA to ~94°C

– DNA becomes denatured

• Annealing – Add primers (short strands of DNA) to separated strands

– Primers combine or hybridize by lowering temp.

• Add DNA polymerase (directs rebuilding of DNA strand) & mixture of free nucleotides.

• Heat to ~55-72 °C

• Repeat process 25-30 times.– This allows over 1 billion copies to be made (32 cycles)

Simple Overview of PCR Amplification

Denature, thenanneal primers (short piecesof DNA to prime DNA synth)

DNA synthesis

Multiple cycles

g of DNA

Short Tandem Repeats (STR)

• Advantages– Better discrimination than RFLP– Faster result time– Low mutation rates

• DNA has sections that repeat bases (2-7) these are used.

• Uses capillary electrophoresis• Visualized as peaks on a graph

What do STRs look like?

4,7 individual

5,5 individual

How STRs Appear As a Result of Analysis

SGM Plus

FSS SGM

CODIS

FBI uses 13 different DNA loci1:53,581,500,000,000,000,000 probability

The Same 13 Locus STR Profile in Different Populations

1 in 0.84 quadrillion (1015) in U.S. Caucasian population (NIST)

1 in 2.46 quadrillion (1015) in U.S. Caucasian population (FBI)*

1 in 1.86 quadrillion (1015) in Canadian Caucasian population*

1 in 16.6 quadrillion (1015) in African American population (NIST)

1 in 17.6 quadrillion (1015) in African American population (FBI)*

1 in 18.0 quadrillion (1015) in U.S. Hispanic population (NIST)

*http://www.csfs.ca/pplus/profiler.htm

1 in 837 trillion

These values are for unrelated individuals assuming no population substructure (using only p2 and 2 pq)

NIST study: Butler, J.M., et al. (2003) Allele frequencies for 15 autosomal STR loci on U.S. Caucasian, African American, and Hispanic populations. J. Forensic Sci. 48(4):908-911. (http://www.cstl.nist.gov/biotech/strbase/NISTpop.htm)

CODIS

• Combined DNA Index System– National DNA I.D. system– Has three levels

• Local

• State

• Federal