Molecular Genetic

Importance of Molecular Genetics

Genetics is playing an important role in the practice of clinical

medicine.
- Medical genetics involves any application of genetics to
medical practice, it thus includes:
Studies of the inheritance of disease in families.
Mapping of disease genes to specific locations on
chromosomes
Analysis of the molecular mechanisms through which genes
cause disease
Diagnosis and treatment of genetic disease (ex. Gene therapy)
 DNA Isolation
DNA isolation: is an extraction process of DNA
from various sources.

The aim: is to separate DNA present in the nucleus

of the cell from other cellular components.
 Application of DNA isolation:

It is needed for genetic analysis which used

for:
1- scientific: use DNA in number of Applications , such
as introduction of DNA into cells & animals or plants for
diagnostic purposes (gene clonining)
2- Medicine: is the most common. To identify point sources
for hospital and community-based outbreaks and to predict
virulence of microorganisms
3- forensic science: needs to recover DNA for
identification of individuals ,( for example rapists, petty
thieves, accident , or war victims) , and paternity
determination.
 Many different methods and technologies are
available for the isolation of genomic DNA.
All methods involve:
A. disruption and lyses of the starting material
followed by
B. Removal of proteins and other contaminants and
finally
C. Recovery of the DNA
 To choice of a method depends on many
factors:

A. The quantity and molecular weight of the

DNA
B. The purity reqired for application
C. The time and expense
 Sample Collection
A- Source: Sample can be isolated from any living or dead
organism
Common sources for DNA isolation include:
Whole blood
Buffy coat
Bone material
Buccal cells
Cultured cells
Amniocytes or amniotic fluid
Sputum, urine, CSF, or other body fluids
 Sample Collection
B. Sample age:
May be fresh or has been stored .
Stored sample can come from:
Archived tissue samples ,
Frozen blood or tissue (biopsy
material) ,
Exhumed bones or tissues &
Ancient human sample.
Dried blood spots
 DNA Purification & Quantification

Separating DNA from other cellular components such as

proteins, lipids, RNA, etc.
Avoiding fragmentation of the long DNA molecules by
mechanical shearing or the action of endogenous
nucleases
Effectively inactivating endogenous nucleases (DNase
enzymes) and preventing them from digesting the
genomic DNA is a key early step in the purification
process. DNases can usually be inactivated by use of
heat or chelating agents.
 Key Steps
Lysis of the cells
Removal of contaminants
includes
Proteins
RNA
Other macromolecules
Concentration of purified
DNA
Use Detergent to solubilize the membrane lipid.
 2. Separate DNA From Crude Lysate

DNA must be separated from proteins and cellular debris.

Separation Methods
a) Organic extraction
b) Salting out
 a) Separation by Organic Extraction
Traditionally, phenol: chloroform is used to extract DNA.
When phenol is mixed with the cell lysate, two phases
form. DNA partitions to the (upper) aqueous phase,
denatured proteins partition to the (lower) organic phase.
Phenol: Denatures proteins and solubilizes denatured
proteins
 b) Separation by Salting Out

At high salt concentration, proteins are dehydrated,

lose solubility and precipitate.
Usually sodium chloride, potassium acetate or
ammonium acetate are used.
Precipitated proteins are removed by centrifugation
DNA remains in the supernatant.
 Separation by Salting Out

Salting out method:

Cell lysis.
Protein digestion by proteinase enzyme.
Protein precipitation by high salt concentration.
Centrifugation will remove the precipitated proteins.
The supernatant contains the DNA.
DNA is then precipitated by adding ethanol.
The precipitated DNA is resuspended in the desired
buffer.
 Ethanol precipitation:
-Precipitation of DNA: Absolute Ethanol is layered on the top of
concentrated solution of DNA
- Fibers of DNA can be withdrawn with a glass rod
- Washing of DNA
- Desalt DNA: Most salts are soluble in 70% ethanol
 2- Use of Commercial DNA purification kits:

The common lysis solutions contain

A. sodium chloride
B. Trimethamine (also known as tris ) , which is a buffer to
retain constant pH
C. Ethylendiaminetetraacetic (EDTA) , which binds metal
ions
D. Sodium dodecyl sulfate (SDS) which is a detergent .
E. An enzyme used in DNA extraction is protienase K
3- Heat denaturation
Achieved by boiling samples.
Heating of a sample to 100 c releases DNA into the solution
but also denatures it by separating the two strand.
Drawbacks: There are remaining inhibitors in the form of
degraded proteins and other organic compound or ions .
 4- Magnetic beads with DNA binding
capacity
Magnetic beads are coated with DNA antibodies or
silica to bind to DNA.
Samples are lyses & and then treated with proteinase K.
The lysates are then applied to the beads.
Resin is subsequently washed & DNA is eluted of it at 65c
Magnetic beads are separated from the sample on a
magnetic stand.
 Summary of DNA extraction :

There are three basic & two optional steps in a DNA

extraction :
1- Cell lysis , to expose the DNA within .
2- removing membrane lipids by adding a
detergents or surfactants .
3- removing proteins by adding a protease .
4- removing RNA by adding an Rnase.
5- precipitating the DNA with alcohol- usually ice
cold ethanol. In these alcohols , DNA strand will
aggregate together, giving a pellet upon centrifugation .
This step also removes alcohol- soluble salt.
 DNA Extraction & Purification:
Evaluation
DNA concentration can be determined by measuring the
intensity of absorbance with a spectrophotometers &
comparing to a standard curve of known DNA
concentration.
Measuring the intensity of absorbance of the DNA solution
at wavelength 260nm & 280nm is used as a measure of
DNA purity
DNA purity: A260/A280 ratio: 1.7 1.9
DNA concentration (g/ml): A260 X 50
DNA yield:
DNA conc. X Total volume of DNA solution
Spectrophotometers
Measurement of DNA purity
Checking for Degradation DNA
Running your sample through an agarose gel is a
common method for examining the extent of DNA
degradation. Good quality DNA should migrate as a high
molecular weight band, with little or no evidence of
smearing.
DNA absorbs UV light at 260 &280 nm & aromatic
proteins absorb UV light at 280 nm Apure sample of
DNA has the 260/280 ratio at 1.8 & is relatively free
from protein contamination.
A DNA preparation that is contaminated with protein
will have a 260/280 ratio lower than 1.8
Agarose gel
Gell Electrophoresis
Vertical electrophoresis
Gell Electrophoresis
 What are some of the DNA technologies
used in forensic investigations?

Restriction Fragment Length Polymorphism

(RFLP)
PCR Analysis
STR Analysis
Mitochondrial DNA Analysis
Y-Chromosome Analysis
Repetitive DNA in the Human Genome
Less than 2% codes for Proteins
50% of the genome contains repeated
sequences
No apparent function
Recombination?
Formation of new genes?

Types of repeated DNA

Tandomly repeated
Telomeres
Satellite (VNTRs)
Minisatellite (STRs)
Interspersed repetitive DNA
SINES (Alu sequences)
LINES
Transposable elements
 DNA in the Cell

Chromosome

cell nucleus

double-stranded
DNA molecule Target Region for PCR

Individual
nucleotides
 What are STRs?

Short Tandem Repeats (STR) are repetitive

sequences:
Tetranucleotide: AAAG AAAG AAAG AAAG
Trinucleotide: CTT CTT CTT CTT CTT
Dinucleotide: AG AG AG AG AG AG
Tetranucleotides are favored in human identity
Good balance of ease of interpretation and variability
found in nature
 D18S51 D18
Chromosomal location FL 18q21.3

1 aattgagcnc aggagtttaa gaccagcctg ggtaacacag tgagacccct gtctctaca

61 aaaaatacaa aaatnagttg ggcatggtgg cacgtgcctg tagtctcagc
tacttgcagg 121 gctgaggcag gaggagttct tgagcccaga aggttaaggc tgcagtgagc
catgttcatg 181 ccactgcact tcactctgag tgacaaattg agaccttgtc
tcagaaagaa agaaagaaag 241 aaagaaagaa agaaagaaag aaagaaagaa
agaaagaaag aaaaagagag ggaaagaaag 301 agaaanagna aanaaatagt agcaactgtt
attgtaagac atctccacac accagagaag 361 ttaattttaa ttttaacatg ttaagaacag
agagaagcca acatgtccac cttaggctga 421 cggtttgttt atttgtgttg ttgctggtag
tcgggtttgt tatttttaaa gtagcttatc 481 caatacttca ttaacaattt cagtaagtta
tttcatcttt caacataaat acgnacaagg 541 atttcttctg gtcaagacca aactaatatt
agtccatagt aggagctaat actatcacat 601 ttactaagta ttctatttgc aatttgactg
tagcccatag ccttttgtcg gctaaagtga 661 gcttaatgct gatcgactct agag

The repeat sequence is aaga this particular individual has 14 repeats

 The locus is where its at

Locusthe physical Both chromosomes of a

position of an STR and its homologous pair contain
associated flanking this locus
sequence

The allele contained on either chromosome can be the same

or different lengths (homozygous or heterozygous)
 Chromosome Spread showing the positions of the amplified loci in PowerPlex16
TPOX
The PowerPlex D3S1358 D5S818
FGA
16 System
amplifies 16 loci. CSF1PO

D7S820 D8S1179 THO1 vWA Amelogenin

D13S317 Penta E D16S539 D18S51

D21S11 Amelogenin
Penta D
 Short Tandem Repeats (STRs)
AATG

~ ~
7 repeats
~ ~
8 repeats
Repeat region is variable (polymorphic)
Each variant is referred to as an allele
Flanking region is constant
KEY: Alleles are distinguished by length

Homozygote = both alleles are the same length

Heterozygote = alleles differ and can be resolved from one another
 DNA Amplification with PCR

5 3
5 3
Starting DNA
Template
3 5
3 5

Separate strands
Forward primer (denature)
5 3

Add primers
3 5
(anneal)
Reverse primer

5 3

Make copies
(extend primers)
3 5
 Exponential Amplification with PCR

Original DNA target region

Thermal cycle

In 32 cycles at 100% efficiency, 1.07

billion copies of amplicon are made.
http://www.cstl.nist.gov/div831/strbase/
Multiplex PCR

16 Loci Are Copied at Once

Sensitivities to levels less
than 0.5 ng of DNA
Ability to Handle Mixtures
and Degraded Samples
Different Fluorescent Dyes
Used to Distinguish STR
Alleles with Overlapping Size
Ranges
 Separating and Seeing STRs
Electrophoresis
Separates amplification products based on size

Fluorescent detection
Amplification products have a fluorescent label attached
to the primer
Label is seen through excitation via a laser and
corresponding emission captured with a camera

377 310 3130

 Current Forensic STR Multiplexes
PowerPlex 16

100 200 bp 300 bp 400 500 bp 600 bp

bp bp

D3 TH01 D21 D18 Penta E

D5 D13 D7 D16 CSF Penta D

A vWA D8 TPOX FGA

10 20 30 40 50 60
0 0 0 0 0 0
 ILS600 Size Standard

100 200 300 400 500 600

A sizing standard is used in all samples and allelic ladders

The known standard is used to determine the size of the allelic ladders
and the unknown samples
Allelic Ladder
Allele Calls
Discrimination power through multiplexing
Allele
possibilities

Hypothetical
likelihood of
occurrence
1 locus: 1 in 18 1 1 1 1
2 loci: 1 in 360
3 loci: 1 in 18000 in X in X in X in
4 loci: 1 in 792000 18 20 50 44
9 loci: ~1 in 1010
Sample
16 loci: ~1 in 1017
Genotype
Current World Pop:
~6.3 billion
 DATA ANALYSIS

Controls
Negative control devoid of amplification products
Compare positive control 9947a with locus-specific ladder

STR Allelic Ladders

Comparison with samples allows precise assignment of
alleles
Fluorescent Ladder (CXR)
Internal Size Standard
 Human Identity Testing
Applications
Forensic cases: matching suspect with evidence
Paternity testing: identifying father
Convicted felon DNA databases
Missing persons investigations
Mass disasters -- putting pieces back together
Historical investigations
Military DNA dog tag


Complete STR Profile

DNA from Small Stains/challenging samples

0.1l blood stain on denim

1/5 of eluted material used for amplification
 DNA Use in Forensic Cases

Most are rape cases (>2 out of 3)

Looking for matches between evidence,
victim, and suspect
Must compare DNA profiles
Challenges

Mixtures must be resolved if present

DNA is often degraded
Inhibitors to PCR and sample
contamination are often present
 Steps in DNA Sample Processing

Sample Obtained
from Crime Scene or
Paternity
Investigation
Biology
DNA DNA PCR Amplification
Extraction Quantitation of Multiple STR markers

Technology
Separation and Detection of PCR Sample Genotype
Products Determination
(STR Alleles)

Comparison of Sample
Genetics Generation of Case Report
Genotype to Other Sample with Probability of Random
Results Match

If match occurs, comparison of

DNA profile to population
databases
 Sources of Biological Evidence

Blood
Semen
Saliva
Urine
Hair
Teeth
Bone
Tissue
 DNA extraction
Samples can have extremely small amounts of DNA
Available Technologies for DNA Isolation
Phenol:Chloroform Extraction (Homebrew)
Chelex (ReadyAmp)
FTA Paper
Qiagen
DNA IQ System
DNA IQ Reference Sample Kit for Maxwell 16
 DNA Quantitation
Forensic labs in the US are required by law to
quantitate the amount of Human DNA in crime
scene samples
How might this be done?
 Regulation of Labs
Forensic
FBI
Standards for Combined DNA Index System (CODIS) labs
http://www.fbi.gov/hq/lab/codis/index1.htm
The Scientific Working Group for DNA Analysis
Methods(SWGDAM) publishes guidelines
Paternity
American Association of Blood Banks (AABB)
http://www.aabb.org
PowerPlex 16
Statistical Analysis
Analysis based on
population statistics and
data
Probability that the
evidence matches the
suspect
 References and resources
http://www.cstl.nist.gov/biotech/strbase/intro.htm
(some information in this presentation is from this ppt)
http://www.promega.com/applications/hmnid/
(Promega Human identity testing products)
http://www.promega.com/profiles/ (Profiles in DNA)
http://journalsip.astm.org/JOURNALS/FORENSIC/jofs_
home.html (Journal of Forensic Science)
http://appliedbiosystems.com Supplier of Human
identification systems