PRTICA FORENSE III

Extrao de DNA das amostras:

1) salting out modificado
2)NaOH
3)orgnica
4)FTA

1) MILLER SA, DYKES DD, POLESKY HF. 1988. A simple salting out procedure for extracting
DNA from human nucleated cells. Nucleic Acids Research, 6:1215.
2) RICHARDS B, SKOLETSKY J, SCHUBER AP et al. 1993. Multiplex PCR amplification from
the CFTR gene using DNA prepared from bucal brushes/swabs. Human Molecular Genetics,
2:159-163.
3) PCR-based typing protocols FBI Laboratory, publicado em 07/01/1996.
4) FTA Protocols, Whatman, publicado em 2002.
 Commercial STR Kits
Kit Contents:
Allelic Ladders for Genotyping
PCR Component Mix
Primer Mix
Positive Control DNA Sample

Custo: ~ R$ 12.000,00

Fornecedores: Applied Biosystems and Promega Corporation

Amplificao das Amostras:

AmpFlSTR IdentifilerTM

COLLINS PJ, HENNESSY LK, LEIBELT CS, ROBY RK,

REEDER DJ, FOXALL PA. 2004. Developmental
validation of a single-tube amplification of the 13 CODIS
STR loci, D2S1338, D19S433, and amelogenin: the
AmpFlSTR Identifiler PCR Amplification Kit. Journal of
Forensic Sciences, Nov; 49(6):1265-77.
22.09.10
6 29,70
1) 6 14,10
6 1,20

S1AQSV
S1ARM
S1ARS
C+
C-
 PCR multiplex

Mais de 10 marcadores podem ser

genotipados ao mesmo tempo

1 ng de DNA ou menos

Diferentes fluorforos (cores)

podem distinguir alelos de STR
que se sobrepem em tamanho

$20-$25 per rxn in lab. $150 incl labor. Cost for forensic up to $1000.
 Amplification
(making copies)

Solution

DNA
 COMPONENTES - Amplificao STRs (kits)

DNA contendo seqncia molde

Tampo + mistura dos 4 dNTPs (T, A, C, G)
Primers (marcados com molcula fluorescente)
DNA polimerase Taq DNA polimerase

Termociclador comanda tempo e C

 DENATURE
Step one of a single cycle

A T A T
G C G C
A T A T
T A T A
A T A T
G C G C

Heat
 ANNEAL
Step two of a single cycle

A G A
A T G
T C T A T C T A T A T
C T
 EXTEND
Step three of a single cycle

A G T G
C A T
T
G GA
T
A G A
A T G A T
A G
T C T A T C T A
T C T A T
 Amplification

28 Cycles

1 Cycle
2 Cycles
3 Cycles
DNA
4 Cycles

5 Cycles
 Analysis of amplified DNA

DNA
Profile

Amplified
DNA
 Genotipagem das Amostras:

Sequenciador ABI PRISMTM 310 Genetic Analyzer, com

auxlio dos softwares ABI Prism 310 Collection, 310
GeneScan v.3.1.2 e Genotyper v.2.5.2
Amostras no Autosampler
Close-up of ABI Prism 310 Sample Loading Area

Electrode
Capillary

Sample Vials

Autosampler Tray
See Technology section for more information on CE
 Eletroforese

Injeo
Eletrocintica

Deteco
ABI 3100 Array Detection
Estrutura do Trabalho entrega e apresentao no dia 29/setembro

1. Introduo: - histrico do caso

- fundamentao terica e marcadores utilizados

2. Objetivos
3. Materiais
4. Mtodos: - coleta
- extrao
- amplificao
- genotipagem
5. Resultados
6. Concluso