PCR Based VNTR human DNA

Question 1

What is polymorphic DNA?

Answer 1

Refers to chromosomal regions that vary widely from one individual to the next

Examination of several of these regions within the genomic DNA obtained from an individual can be used to generate a “DNA fingerprint” for that individual

Question 2

What a VNTR?

Answer 2

A region that is variably composed of a 15-70 base pair sequence tandemly arranged heal to tail, typically repeated 5-100 times

Question 3

What is an STR?

Answer 3

An STR is similar to a VNTR except that the repeated unit is only 2-4 nucleotides in length

By examining several different VNTRs or STRs from the same individual, investigators obtain a unique DNA fingerprint for that individual which is unlike that of any other person (except for an identical twin)

Question 4

When did Did DNA fingerprint start?

Answer 4

In the UK in 1984, following the pioneering work of Dr. Alex Jeffreys at the. University of Leicester

-The first US conviction occurred on November 6, 1987 in Orlando, FL

In 1990, the Federal Buraeu of Investigation (FBI) established the Combined DNA Index System(CODIS), a system which allows comparison of crime scene DNA to DNA profiles in a convicted offender and a forensic (crime scene) index
-A match of crime scene DNA to a profile in the convicted offender index indicates a suspect for the crime

-Whereas a match of crime scene DNA to the forensic index (a different crime scene) indicates a serial offender

Question 5

How are DNA Polymorphisms used in forensic science?

Answer 5

Polymorphisms used in forensic science, for example in determining:

• paternity/maternity
• kinship
• identification of human remains
• in the field of criminal forensics

Question 6

Briefly describe how forensics uses Polymorphisms

Answer 6

Any match between the crime scene DNA and a suspects DNA at a single locus doesn’t prove guilt, nor does it rule out innocence. Therefore, multiple loci are tested and evaluated

For most applications, law enforcement agencies now use STRs as they are more easily amplified and thus require less starting DNA

Question 7

How is forensic DNA fingerprinting done?

Answer 7

Collect sample: human tissue (blood, hair, skin, body fluids) from the crime scene or victim.

Treat sample (e.g. with a detergent) to lyse the cell membranes and obtain the DNA for further analysis.

Amplify (by Polymerase chain reaction)-specific highly polymorphic DNA regions which vary in length from individual to individual and fall into two categories:

1. Variable Number of Tandem Repeats(VNTRs)
2. Short tandem repeats (STRs)

Separate and examine the amplified DNA segments by Agarose gel electrophoresis. Bands of crime scene DNA to DNA from suspect/s are compared

Question 8

What is a DS180?

Answer 8

A VNTR, present on chromosome 1 and contains a 16-nucleotide sequence which is variably repeated between 16 and 40 times

An individual who is homozygous for the DS180 genotype will have equal repeat numbers on both homologues of chromosome 1, displaying a single PCR product following gel analysis

More commonly, a person will be heterozygous, with differing DS180 repeat numbers

Question 9

What will amplification of DNA from Heterozygous induvuduals cause?

Answer 9

Result in two distinct products

The enormous utility of PCR based on its ease of use and its ability to amplify DNA

Question 10

Describe PCR

Answer 10

The PCR amplification uses an enzyme known as Taq polymerase
-This enzyme, originally purified from a bacterium that inhabits hot springs, is stable at very high temperatures

Also included in the PCR reaction mixture are two synthetic oligonucleotides (15-30 nucleotides) known as “primers “ abs the extracted DNA is known as the “template”

The region of DNA to be amplified is known as the “target”

Question 11

What are the steps in PCR?

Answer 11

In the first step of the PCR reaction, the template complementary DNA strands are separated(denatured) from each other at 94 degrees Celsius, while the Taq polymerase remain stable

In the second step, known as annealing, the sample is cooled to an intermediate temperature (usually 40-65 degrees Celsius) to allow hybridization of the two primers to the strands, one to each of the two strands, one to each of the two strands of the target DNA sequence

In the third step, known as extension (also sometimes called DNA synthesis), the temperature is raised to 72 degrees Celsius and the Taq polymerase adds nucleotides to the primers to complete the synthesis of the new complementary strands

Question 12

Summarize the steps of PCR

Answer 12

Denaturation, annealing, extension-constitute one PCR cycle. This process is typically repeated for 30-40 cycles, amplifying the target sequence exponentially

Question 13

PCR is performed in a…

Answer 13

Thermal cycler, an instrument that is programmed to rapidl6 heat, cool, and maintain samples at designated temperatures for varying amounts of time

Question 14

What are the experimental objectives?

Answer 14

The objectives of this experiment are:

• to isolate human DNA
• compare DNA Polymorphisms between individuals
  
  • by PCR amplification and
  • gel electrophoresis
  • identify Polymorphisms in the D1S80 region of chromosome 1

In this experiment, each student will

- 1. Extract his/her DNA from hair or cheek cells
- 2. Amplify DNA at the D1S80 locus by PCR
- Examine the PCR products on agarose gels

Question 15

Explain the isolation of DNA from human cheek cells

Answer 15

• A sufficient volume of cells is required To ensure that there is enough DNA to give positive DNA fingerprinting results
• to maximize success, carefully read and follow all experimental instructions
• Special caution is required when handling human samples
• use hand gloves
• Contaminated laboratiry waste(saliva solution, cup, pipet, etc.) must be disinfected with 15% bleach solution prior to disposal
• Proper dispose any biological sample- if unsure, ask your instructor

Question 16

How can DNA be isolated from human cheek cells?

Answer 16

1. Label a 1.5 ml screw top microcentrifuge tube and a cup with your lab group and/or initials
2. Rinse your mouth vigorously for 60 seconds using 10 ml saline solution. EXPEL the solution into cup
3. SWIRL the cup gently to resuspend the cells. TRANSFER 1.5 ml of solution into the labelled tube
4. CENTRIFUGE the cell suspension for 2 min at full speed to pellet the cells. POUR off the supernatant, but DO NOT DISTURB THE CELL PELLET! Repeat steps 3 and 4 twice more
5. RESUSPEND the cheek cells in 140 uL lysis buffer by pipetting up and down or by vortexing vigorously
   
   • The lysis solution contains 25mM Tris-HCl pH 8.0 and 5% chelating agent. The chelating agent removes Mg (required by DNA degrading nucleases and DNA polymerases)
6. CAP the tube and place in a water bath float. Incubate the sample in a 55 degrees Celsius water bath for 15 min
7. MIX the sample by vortexing or flicking the tube vigorously fir 20 seconds
8. INCUBATE the sample in a 99 degrees Celsius water bath for 15 min. Be sure to use screw cap tubes when boiling DNA isolation samples
   
   • boiling is required to obtain cell lysis. The DNA is not degraded by boiling and nucleases will not degrade the DNA because the Mg is chelated (trapped)
9. Centrifuge the cellular lysate for 2 minutes at low speed (6000 rpm)
10. TRANSFER 80 uL of the supernatant (DNA) to a clean, labeled microcentrifuge tube. Place tube in ice.
    
    • This is the step prior to the PCR reaction. If any chelex beads (as few as one or two ) are transferred, they can easily chelate (trap) the Mg required by the Taq DNA polymerase as a cofactors for catalysis. any carry-over of chelex to the PCR reaction will not yield results

Question 17

Explain the digestion of Proteinase K

Answer 17

Originally extracted from the fungus Tritirachium album
-Named due to its ability to cleave hair keratin

Many proteinases only cleave after a specific amino acid

- This leads to the production of large fragments
- Proteinase K is relatively non-specific, therefore leaving very small fragments

Is active over a wide range of temperature(Optimum activity at 50 to 55 degrees Celsius)

• Is active in the presence of a wide range of additives including SDS & EDTA
• Proteinase K rapidly inactivates DNases and RNases in most microbial or mammalian cells

Question 18

What are the steps of PCR Amplification

Answer 18

1. Label the tubes (x3), each containing the PCR reaction pellet with your initials or group number.
   
   • The PCR reaction pellet contains Taq polymerase, the four deoxytriphosphates, Mg2+ and buffer
   • Sample volumes are small. It is important to quick spin the tube contents in a microcentrifuge to obtain sufficient volume for pipeting. Spin samples for 10-20 seconds at maximum speed
2. Tap the reaction tubes(x3) to assure the reaction pellet is at the bottom of each tube.

3. Add the following to the pellet in each tube- D1S80 primer solution 20.0 uL 
Cell DNA (supernatant) 5.0 uL 

4. Gently mix each PCR reaction tube (x3) and quick spin it in a microcentrifu to collect all the sample at the bottom of the tube. Make sure the PCR reaction pellet is completely dissolved.
5. Label a 0.2 ml PCR tube with your initials or group number. X3
6. Carefully transfer the entire contents of your PCR reaction tube to the 0.2 ml PCR tube x3
7. Once you have completed the set up of the PCR reaction your instruction will bring you to the thermal cycler machine. You will place your tubes (x3) in the machine
8. The PCR parameters are as follows:
   Initial denaturation: 94 degrees Celsius for 4 min.

35 cycles @: 94 degrees Celsius for 30 sec. ,65 degrees Celsius for 30 sec., and 72 degrees Celsius for 30 sec

Final extension: 72 degrees Celsius for 4 min

Question 19

How long does PCR amplification take?

Answer 19

The PCR program will typically run for 1-2 hours. After the PCR program is completed 5 uL of gel loading dye will be added to each samples before stor8ng them in the freezer

Agarose gel electrophoresis

Immediately prior to the start of the second part of lab #6, the samples and the 200 bp DNA ladder will be heated in a water bath at 50 degrees Celsius for 2 minutes

Several class groups will load samples on the same gel si that a comparison can be made between individuals

Question 20

What are the steps of agarose gel electrophoresis?

Answer 20

1. Carefully rem9ve the rubber dams and comb
2. Place the agarose gel in the electrophoresis tank
3. Add electrophoresis buffer to the tank until the gel is completely submerged
4. Load 30 uL of your sample in an unused well (2-6) and note the well number on the sheet pr9vided
5. Place the lid on the tank-match terminals by color. Look for bubbles forming at each electrode to confirm electricity flow through