C4 Flashcards
What is DNA polymorphism
- Variation of DNA seq or length common in given population
- Used for personal identification
- Most are neutral but some susceptible to diseases
Polymorphism appears at different levels of
- Phenotype polymorphism
- Protein polymorphism
- Genetic polymorphism
What causes DNA polymorphism
- Variation in fragment length pattern produce after digestion DNA with restriction enzyme
- Variation in size of DNA (after PCR)
- Variation in DNA sequence
Types of DNA polymorphism
- RFLP
- VNTR
- SSR
- SNP
Explain Restrictions Fragment Length Polymorphism (RFLP)
- Segment of DNA digested with restriction enzyme E
- Segment can be identified in Southern blot or PCR
Explain the mechanism of enzyme E in RFLP
- 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
RFLP is
Biallelic, give 2 fragment size depend on whether the polymorphism restriction depends whether polymorphic restriction fragment site absent or present
Explain variable number of tandem repeat (VNTR)
- 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
Why VNTR more informatics
Because of their greater intrinsic variability since there is a greater chance that heterozygous patterns will be detected in one locus
Example of VNTR
Minisatellites
Characteristics of SSR/STR
- 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
Explain SSR
- Important to track inheritance in family & DNA fingerprinting
- Amplification of DNA contain SSR produce fragments of variable size
Characteristics of SNP
- Known as SNV
- Most common
- Variations at single nucleotide
- Biallelic & polymorphic
Define mutation
DNA gene is damaged or changed in such a way alter the genetic message carried by that gene
Define mutagen
Agent of substance that can bring permanent alterations to the physical composition of DNA gene
Types of mutation
- Silent
- Missense
- Nonsense
- Insertion
- Deletion
Characteristics of SNP
- Mutation that changes one base pair
- Very common
- Most are junk DNA
Why SNP important
- As genetic marker
- Allow to rapidly & cheaply deduced genotype individuals
- Some SNP directly associated with diseases
Difference between mutation & polymorphism
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
Detections of gene mutation
- Biochemical methods
- Nucleic acid analysis
- Hybridisation based method
Principle of SSCP
- 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
Procedure in SSCP
- DNA sample preparation
- Denaturation
- Annealing
- Electrophoresis
- Visualisation
- Analysis
Explain procedure of SSCP
- 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
Pro & cons SSCP
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
Application of SSCP
- Genetic research: identify DNA sequence variation
- Forensic DNA
- Molecular microbiology: identify related bacteria species
Principle of Denaturing Gradient Gel Electrophoresis (DGGE)
- 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
Procedure of DDGE
- 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
Pros & Cons of DGGE
Pros
- High sensitivity
- High resolution
- Not effective by environmental factor
- Fast & cheap
Cons
- Long running time
- Require special equipment
- Require great experience
Application of DGGE
- To detect non-RFLP polymorphism
- Food microbiology: detect bacteria strain
- Medical research: identify DNA sequence variant associated with disease
- To type alleles of polymorphism
Comparison between DGGE & SSCP
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
Principle of molecular beacon
- 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
Procedure of molecular beacon
- 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
