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SFN 316 Forensic Molecular Biology > Chapter 3 > Flashcards

Chapter 3 Flashcards

1
Q

The Principles of RFLP Testing

A
  1. Cut the DNA with “biological scissors” (restriction enzymes)
  2. Separate fragments of differing length by gel electrophoresis
  3. Detect length-based differences (polymorphisms) in DNA fragments of interest
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2
Q

What does RFLP stand for?

A

Restriction Fragment Length Polymorphism

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3
Q

VNTRs

A

Variable Number Tandem Repeats

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4
Q

What are VNTRs?

A

9-75 base pair sequence of DNA that is repeated back to back a different number of times in different individuals

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5
Q

Nucleases

A

Enzymes that degrade nucleic acids

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6
Q

What do nucleases do?

A

Hydrolytically cleave DNA at the phosphate (PO4-) group

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7
Q

Micrococcal Nucleases

A

Cleave DNA that is not protected by proteins, such as linker DNA between nucleosomes

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8
Q

Endonuclease

A

Cleaves DNA within a chain of DNA

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9
Q

Exonuclease

A

Cleaves DNA one nucleotide at a time from the end of a chain of DNA

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10
Q

Restriction endonuclease (AKA Restriction Enzymes)

A

Recognize specific sequences of DNA and cleave both strands of DNA at sites internal to the molecule

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11
Q

Can restriction enzymes cleave single-stranded DNA?

A

No, it has to be double-stranded

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12
Q

Where do restriction enzymes originate from?

A

Bacteria evolved REs to destroy DNA from invading viruses or bacteriophages

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13
Q

What sequence is recognized by restriction enzymes?

A

A palindrome sequence, which is where the complementary strands of DNA read the same in both directions

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14
Q

Recognition site

A

4-8 base pairs long
Specific sequence of DNA that a particular RE recognizes for cleavage

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15
Q

Restriction site

A

The actual bonds within the recognition site that are cleaved by the RE; where the enzymes cut

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16
Q

Blunt ends

A

DNA that has been cleaved is double-stranded
Straight cut

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17
Q

Sticky ends

A

DNA that has been cleaved has one or more unbound nucleotide at the cut site; uneven cut with cohesive ends

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18
Q

Digestion

A

Cutting high molecular weight DNA with a RE

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19
Q

Partial Digestion

A

The RE cuts the DNA at some sites, but not all the sites it is supposed to

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20
Q

Post-restriction Test Gel

A

An agarose gel used to test for complete digestion

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21
Q

What does a streak in a post-restriction test gel indicate?

A

Digested DNA, since the DNA has different sizes

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22
Q

What does a distinct band in a post-restriction test gel indicate?

A

At a high molecular weight area, it indicates partial digest

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23
Q

Electrophoresis

A

Separating DNA or proteins based on size using an electric current

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24
Q

Southern Blot

A

Transfer of DNA from an agarose gel to a nylon membrane through capillary action

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25
Q

What binds to the nylon membrane in the Southern Blot method?

A

Only single-stranded DNA will bind

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26
Q

Why do we want to examine different loci?

A

To increase the power of discrimination

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27
Q

Denaturation of DNA

A

Process of separating complementary strands of double stranded DNA into single strands of DNA

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28
Q

How can the denaturation of DNA be performed?

A
  1. Heat
  2. Salt
  3. pH extremes
  4. Chaotropic agents
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29
Q

What does denaturation do to the DNA?

A

Destabilizes hydrogen bonds and hydrophobic interactions

30
Q

What is used in Southern Blot for chemiluminescent detection?

31
Q

What is used in Southern Blot for P32 detection?

32
Q

Why is a Southern Blot gel placed into UV light?

A

DNA is attached to the nylon membrane, but not covalently linked. The UV light fixes the DNA to the membrane.

33
Q

Probe

A

Short single-stranded sequence of DNA that is complementary to an area of DNA that is being examined

34
Q

What is a probe generally labeled with?

A
  1. Radioactive P32
  2. Chemiluminescent tag - break down of a chemical to give off light
  3. Biotin - color development
35
Q

Why is the prehydridization step necessary?

A

The probe being used is single stranded and could bind to the nylon membrane

36
Q

What does the prehybridization step do?

A

Blocks areas of the membrane where DNA is not bound so the probe will not bind to the nylon membrane, but rather to the complementary sequence

37
Q

What conditions are used for the prehybridization step?

A

Performed at a temperature that is optimum for the probe to bind to its complementary strand
65 degrees C for P32
55 degrees C for Chemiluminescence

38
Q

Stringency Washes

A

Washes away unbound probe and non-specifically bound probe; being strict with the hybrid permitted to form

39
Q

Visualization of DNA on membrane

A

Membrane bound with the probe is taped to two pieces of film and allowed to develop

40
Q

How long does it take for the film to develop in visualization?

A

The film is exposed to the membrane for up to two weeks (P32) or several hours (Chemi)

41
Q

What is the film called in the visualization of DNA using radioactive probes?

42
Q

Chemiluminescent probes react with Lumiphos

A

Will give off light that turns a portion of the film black; film referred to as a lumograph

43
Q

Why are multi-locus probe RFLP results harder to interpret?

A

They contain complex patterns

44
Q

Why are single-locus probes better for forensic samples containing mixtures?

A

There are only 2 bands per individual

45
Q

Nonmenclature for autoradiograms

A

Lanes numbered sequentially from left to right, DNA described as bands

46
Q

Stripping of probes

A

Probe is stripped from membrane so another probe can be hybridized to the membrane

47
Q

What happens if the probe is not completely stripped from the membrane?

A

Bands will be visible on later probes

48
Q

Bioimaging systems and sizing of bands

A

Films are scanned into a computer and computer-assisted imaging is used to dram lanes and place bands; ladder with bands of known length is used to determine the size of sample bands

49
Q

Advantages of RFLP with Single Locus VNTR Probes

A
  1. Excellent powers of discrimination
  2. Large number of alleles at each locus, which facilitates mixed-sample analysis
50
Q

Disadvantages of RFLP with Single Locus VNTR Probes

A
  1. Limited sensitivity (>50ng to 500ng needed)
  2. Time consuming process (days to weeks) that cannot be automated
  3. Not suitable with degraded DNA samples due to high molecular weight needed
  4. Essentially continuous allele sizes which required grouping alleles into bins. Binning introduces statistical complications
  5. Limited number of validated loci
51
Q

Recombinant DNA

A

Cutting up DNA molecules and splicing together fragments from more than one organism

52
Q

Transformation

A

The genetic alteration of a bacterial cell resulting from the transfer of foreign DNA

53
Q

Cloning vector

A

A plasmid or phage that is used to carry inserted foreign DNA for the purposes of producing more material or protein product

54
Q

Plasmid

A

A linear or circular double-stranded DNA that is capable of replicating independently of the chromosomal DNA

55
Q

Why are plasmids important in certain bacteria?

A

Because they code for proteins, especially enzymes, which can confer resistance to antibiotics

56
Q

DNA ligase

A

An enzyme that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond

57
Q

Polymarker-DQ Alpha (PM/DQA1)

A

5ng of DNA amplified
Long strips of nylon membrane are spotted with different sequences of DNA (alleles) found at a particular locus

58
Q

Reverse Dot Blot

A

Probe bound to membrane (SSO - Sequence Specific Oligonucleotide)
Sample washed over membrane
Amplified product is labeled with a biotin tag

59
Q

Reverse Dot Blot Detection

A

Relies on the hybridization of sample PCR products to test allele dots

60
Q

How are results from reverse dot blot interpreted?

A

A positive result for an allele of interest will be indicated by a blue color change

61
Q

Advantages of Reverse Dot Blot

A
  1. Fast, simple method (compared to RFLP)
  2. Capable of analyzing small or degraded samples because it uses PCR
  3. No instrumentation needed after PCR
62
Q

Limitations of Reverse Dot Blot

A
  1. Poor power of discrimination with only 6 loci developed each containing only a few alleles
  2. Mixture interpretation is difficult with limited number of alleles per locus
63
Q

D1S80

A

16 base pair repeat sequence, AmpFLP (Amplified fragment length polymorphism)
PCR products in 400-800bp range

64
Q

How does D1S80 work?

A

Separation on a polyacrylamide gel ,which is detected with silver staining

65
Q

What is D1S80 usually coupled with?

A

Amelogenin, to facilitate sex-typing

66
Q

Advantages of D1S80

A
  1. Improved sensitivity compared to RFLP because it uses PCR
  2. Many alleles which facilitates mixed-sample analysis
  3. Discrete allele calling possible using allelic ladder, which also simplifies statistical interpretation
67
Q

Limitations of D1S80

A
  1. Large allele range, making it difficult to multiplex with other loci and giving rise to preferential amplification of smaller alleles
  2. Poor power of discrimination as a single locus
  3. Allele dropout seen with highly degraded DNA
  4. Gel separation and silver-stain detection not amenable to automation or high-throughout sample processing
68
Q

Advantages of Silver-Stained STRs

A
  1. Sensitive due to PCR
  2. Relatively rapid process (1-2 days)
  3. Works well with degraded DNA samples since shorter fragments of DNA can be analyzed (compared to D1S80)
  4. Lower start-up cost compared to fluorescent STRs
69
Q

Limitations of Silver-Stained STRs

A
  1. Because only a single ‘color’ channel is available, multiplex amplification and detection is limited to 3 to 4 loci
  2. Both strands of DNA are detected leading to double hands with some loci that can complicate interpretation
70
Q

Advantages of PCR Methods

A
  1. Very small amounts of DNA templated may be used - even as little as from a single cell
  2. DNA degraded to fragments only a few base pairs in length can serve as effective templates for amplification
  3. Large numbers of copies of specific DNA sequences can be amplified simultaneously with multiplex PCR reactions
  4. Contaminant DNA, such as from fungal and bacterial sources, will not amplify because human specific primers are used
  5. Commercial kits are not available for easy PCR reaction setup and amplification
71
Q

Potential pitfalls with PCR methods

A
  1. The target DNA template may not amplify due to the presence of PCR inhibitors in the extracted DNA
  2. Amplification may fail due to sequence mutations in the primer-binding region of the genomic DNA template - something often referred to as a ‘null allele’
  3. Contamination from other human DNA sources besides the forensic evidence at hand or previously amplified DNA samples is possible without the use of careful lab technique and validated protocols

SFN 316 Forensic Molecular Biology (4 decks)

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