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MIC661:MOLECULAR DIAGNOSTIC > C4 > Flashcards

C4 Flashcards

1
Q

What is DNA polymorphism

A
  • Variation of DNA seq or length common in given population
  • Used for personal identification
  • Most are neutral but some susceptible to diseases
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2
Q

Polymorphism appears at different levels of

A
  • Phenotype polymorphism
  • Protein polymorphism
  • Genetic polymorphism
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3
Q

What causes DNA polymorphism

A
  • Variation in fragment length pattern produce after digestion DNA with restriction enzyme
  • Variation in size of DNA (after PCR)
  • Variation in DNA sequence
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4
Q

Types of DNA polymorphism

A
  • RFLP
  • VNTR
  • SSR
  • SNP
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5
Q

Explain Restrictions Fragment Length Polymorphism (RFLP)

A
  • Segment of DNA digested with restriction enzyme E
  • Segment can be identified in Southern blot or PCR
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6
Q

Explain the mechanism of enzyme E in RFLP

A
  • Result from point mutation affect single restriction enzyme recognition site (E) that are either present or absent
  • Absent: Enzyme E digest DNA at 2 outside E site
  • Present: Enzyme E digest DNA at E & 2 outer E site
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7
Q

RFLP is

A

Biallelic, give 2 fragment size depend on whether the polymorphism restriction depends whether polymorphic restriction fragment site absent or present

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8
Q

Explain variable number of tandem repeat (VNTR)

A
  • Has the potential to more polymorphic due to changes in E specific restriction fragments brought by insertion of variable number of repeat unit
  • More polymorphic fragments generated
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9
Q

Why VNTR more informatics

A

Because of their greater intrinsic variability since there is a greater chance that heterozygous patterns will be detected in one locus

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10
Q

Example of VNTR

A

Minisatellites

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11
Q

Characteristics of SSR/STR

A
  • Sequence of 2/ more DNA base that are repeated numerous time in head to tail manner
  • Present in non coding DNA
  • Detect using PCR
  • Smaller than RFLP/VNTR
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12
Q

Explain SSR

A
  • Important to track inheritance in family & DNA fingerprinting
  • Amplification of DNA contain SSR produce fragments of variable size
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13
Q

Characteristics of SNP

A
  • Known as SNV
  • Most common
  • Variations at single nucleotide
  • Biallelic & polymorphic
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14
Q

Define mutation

A

DNA gene is damaged or changed in such a way alter the genetic message carried by that gene

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15
Q

Define mutagen

A

Agent of substance that can bring permanent alterations to the physical composition of DNA gene

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16
Q

Types of mutation

A
  • Silent
  • Missense
  • Nonsense
  • Insertion
  • Deletion
17
Q

Characteristics of SNP

A
  • Mutation that changes one base pair
  • Very common
  • Most are junk DNA
18
Q

Why SNP important

A
  • As genetic marker
  • Allow to rapidly & cheaply deduced genotype individuals
  • Some SNP directly associated with diseases
19
Q

Difference between mutation & polymorphism

A

Mutation
- caused by various factor
- uncommon & occur randomly
- various effect (harmful, beneficial, neutral)
- Persist & can be passed on

Polymorphism
- naturally occur
- widespread & not rare
- Neutral / minimal effect
- Persist

20
Q

Detections of gene mutation

A
  • Biochemical methods
  • Nucleic acid analysis
  • Hybridisation based method
21
Q

Principle of SSCP

A
  • Separate ssDNA/RNA based on mutation related conformational differences
  • Detects sequence variation by electrophoretic mobility differences
  • Alteration can cause ssDNA to migrate differently under nondenaturing conditions
22
Q

Procedure in SSCP

A
  • DNA sample preparation
  • Denaturation
  • Annealing
  • Electrophoresis
  • Visualisation
  • Analysis
23
Q

Explain procedure of SSCP

A
  • Prepare DNA fragments by digestion with RE or PCR
  • Denature DNA in alkaline solution
  • Separate DNA fragments by neutral polyacrylamide gel electrophoresis
  • Stain gel (silver/ethidium bromide) or transfer gel on nylon membrane followed by hybridisation of probe
  • Compare mobility of control w unknown DNA fragments
  • DNA sequencing
24
Q

Pro & cons SSCP

A

Pros
- Simple & cheap
- Detect wide range of sequence variation
- Sensitive

Cons
- Limited sensitivity
- Affected by environmental factor (temp & pH)
- Not suit to analyse large DNA fragments

25
Q

Application of SSCP

A
  • Genetic research: identify DNA sequence variation
  • Forensic DNA
  • Molecular microbiology: identify related bacteria species
26
Q

Principle of Denaturing Gradient Gel Electrophoresis (DGGE)

A
  • Use gradient gel with decreasing pore size to increase resolution
  • Fragment with minor, even single nucleotide can be separated
  • PCR + DGGE permit separation of all DNA variants
27
Q

Procedure of DDGE

A
  • Prepare gradient gel with aid of gradient maker at room tempt
  • Place solidified gel into tank contain gel buffer at 60C with recirculation pump turn on
  • Load the sample
  • Electrophoresis run at 65-75V
  • Stop electrophoresis & stain he gel with ethidium bromide
  • Examine gel by UV illumination
  • DNA transfer onto nylon blots
28
Q

Pros & Cons of DGGE

A

Pros
- High sensitivity
- High resolution
- Not effective by environmental factor
- Fast & cheap

Cons
- Long running time
- Require special equipment
- Require great experience

29
Q

Application of DGGE

A
  • To detect non-RFLP polymorphism
  • Food microbiology: detect bacteria strain
  • Medical research: identify DNA sequence variant associated with disease
  • To type alleles of polymorphism
30
Q

Comparison between DGGE & SSCP

A

SSCP
- Generate ssDNA by nuclease digestion
- Use non denaturing PAGE
- 1 band per species
- Not widely accepted

DGGE
- Generate dsDNA with GC clamp
- Use denaturing gradient PAGE
- More than 1 band per species
- Well established

31
Q

Principle of molecular beacon

A
  • Specialised ssDNA/RNA molecules design for molecular & genetics research
  • Act as probes to detect the presence or absent of specific nucleic acid in sequence
  • Based on their ability to emit fluorescence in present of their target sequence
32
Q

Procedure of molecular beacon

A
  • Design & synthesis of molecular beacon sequence
  • Selection of suitable fluorophore & quencher pair to attach to end of MB stem
  • Preparation of hybridisation assay (MB + target + buffer)
  • Amplifies of target sample using real time PCR, RT-PCR, qPCR
  • Detection of fluorescence when MB hybridise with target sequence
  • Analysis to quantify amount of target sequence

MIC661:MOLECULAR DIAGNOSTIC (7 decks)

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