Forensic Bio 2 Exam 2

The use of PCR technology

amplify specific DNA segments creating billions of copies from minimal samples within hours

In the PCR process, the copy number ____ in every cycle

doubles

The three steps in a PCR cycle and associated temperatures

1. Denaturation 94-98 degrees celsius

2. Annealing 50-65 degrees celsius

3. Extension 72 degrees celsius

The use of real time PCR technology

amplification and quantification of DNA and RNA in real time

Structural characteristics of a primer in a PCR reaction

1. single stranded

2. artificially synthesized

3. 15-20 bases long

4. complementary sequence to target

Disadvantages of using the PCR system for forensic human identification

1. contamination issues

2. equipment

3. power of discrimination

Advantages of using the PCR technology for forensic human identification

1. time for analysis

2. amount of DNA

3. quality of DNA

What is a threshold cycle in real time PCR?

The cycle at which a sample’s amplification curve crosses the threshold

What is threshold?

The level of detection or point at which a reaction reaches a fluorescent intensity above the baseline

The name taq-man probe is derived from the game ____

Pac-man

Taqman probe structure and parts

The amount of fluorescence released during a realtime PCR cycle is directly proportional to the amount of ___ present in the sample

DNA

In addition to the PCR components, a _____ is added to the real time PCR reaction

taq-man probe

Spectrophotometric DNA quantitation can be used to detect total human DNA present in a sample

False

What is the formula used to quantitate the total DNA present in a sample by spectrophotometric method?

A260 of 1.0 = 50 micrograms/mL of DNA

In an electrical field, during electrophoresis the DNA molecules migrate towards ____ charge

positive

Three types of electrophoresis

1. Agarose Gel

2. Acrylamide Gel

3. Capillary Electrophoresis

In this electrophoretic separation system, the DNA samples are run horizontally.

Agarose Gel

While using a multiplex PCR kit for human identification, the different size PCR products are separated in an electrophoretic system by ___ and ____

size and fluorescent label detection

The kit name cTTv stands for____

CSF1PO, TPOX, TH01, vWA

For a 15 marker autosomal STR analysis, and one amelogenin marker, how many alleles are amplified?

16

In the Identifier STR analysis kit, that amplifies 15 STR markers are one amelogenin marker, how many primers are included?

32

The identifier STR kit from Applied Biosystems is a ___ color detection system

5

The Promega Powerplex 35GY 8C system is a ___ color detection system

8

The number of loci recommended by CODIS (as of now) for forensic human identification

20

Components of PCR and Real time PCR

PCR

• Template DNA (sample) 1ng

• Primers (2)

• Taq DNA Polymerase

• Buffers (MgCl2/MgSO4)

• dNTP’s

Real Time

• DNA

• Primers

• Taq Polymerase

• Buffers

• dNTP’s

• Taq man probe (measures bases and sequences)

PCR inhibitors from different sources

Blood, Feces, Urine, Tissue

Setting up a PCR reaction in a laminar flow hood will prevent ____

contamination

In a PCR reaction using the multiplex human identification kit, the entire human genome is copied rather than small segments of the human DNA

False

Name any 10 STR markers/loci used for forensic DNA analysis

• vWA

• FGA

• D3S1358

• D8S1179

• D21S11

• D18S51

• D5S818

• D13S317

• D7S820

• CSF1PO

Precautions employed to avoid contamination in a forensic DNA analysis laboratory

1. Mandatory PPE

2. Segregation of pre- and post- amplification rooms

3. Frequent surface decontamination with UV light or bleach

4. Using disposable certified sterile tools

Multiplex PCR saves time and samples

True

The reason to include a reagent blank in DNA extraction and PCR amplification is……

to detect possible contamination in the reagents

In an electropherogram, you see one peak for one locus and two peaks for another locus, why?

Humans inherit two sets of chromosomes, a single locus typically shows one peak (homozygous) or two peaks (heterozygous)

The two major components that supply forensic DNA analysis kits and reagents are…..

Promega Corporation and Thermo Fisher Scientific

What is an amelogenin marker used for?

Determine the sex of a human sample

In the ABI identifier multiplex kit, the fluorescent PCR fragments are generated by

fluorescent primers

Non-human DNA will not interfere with forensic STR marker amplification because:

The PCR primers are designed to be highly specific to human DNA

What are the four different types of detection systems for the amplified STR-alleles?

1. Silver-staining of gels (Acrylamdie)

2. Flatbed scanners (Hitachi FM Bio)

3. Scan fragments as it comes out of gel

4. Capillary electrophoresis

In a multiplex PCR amplification, the two PCR products of the same size from two different loci can be separated in a capillary electrophoretic system by___

dyes

The DNA/PCR product separation system for STR markers should be able to separate fragments that differ by one base pair

True

What is capillary electrophoresis?

analytical technique used to separate DNA based on its charge to mass ratio

Number of capillaries and electrodes in a ABI 310 genetic analyzer

1 capillary and 2 platinum electrodes

In an electropherogram, the big dye blob in the beginning of the raw data is due to

dye terminators

In a capillary electrophoresis the smallest fragments to reach the detection window are

the fastest to arrive

What are two advantages of ABI 310 capillary genetic analyzer?

1. Automated high-resolution electrophoresis

2. Rapid, sensitive analysis of DNA samples

The inner diameter of a capillary used in ABI 310 instruments is

50 microns

The length of the capillary used in ABI 310 instrument is

47 cm

What is a singleplex and what is a multiplex?

Singleplex amplifies a single specific target sequence per reaction and multiplex amplifies two or more sequences simultaneously in a single reaction often labeled with different colored probes.

Number of alleles in autosomal markers versus Y STR markers.

Autosomal STR markers display two alleles per locus and Y-STR markers show one allele

What is the reason to include negative control (no template control) during the PCR set up?

To detect reagent or environment contamination

The number of alleles amplified in a:

• 4 marker autosomal STR system

• 8 marker Y chromosomal STR system

• Promega Powerplex 16 multiplex kit

• ABI Identifier multiplex kit

8, 8, 16, 15

The terms ‘plex’ and ‘filer’ are associated with these two companies

Applied Biosystems and Promega Powerplex

What category does this pattern belong to?

• TH01 (AATG) 5-11

Simple repeat

What category does this repeat pattern belong to?

• (TCTA) (TCTG) 3-4 (TCTA)n

Compound repeat or vWA

What category does this repeat pattern belong to?

•  (TCTA) 4-6 (TCTG) 5-6 (TCT) 3 TA(TCTA)3 TCA(TCTA) 2 TCCATA (TCTA) 8-16 TC

Complex repeat or D21S11