Module 3 Flashcards
Paved the way for genomics
Evaluate genetic variation within and among individuals, species, and higher order taxonomic groups
DNA Marker Technologies
For genetic variation to be useful to geneticists, they
must be:
- heritable
- discernible to the researcher
Genomic sources:
Nuclear genes and Plastid (Mitochondrial) genes
- Usually come in pairs in diploid organisms
- May occur as different alleles in heterozygotes
Nuclear Genes
- Maternally inherited
- Usually more variable than nuclear genes due to rapid mutation
- Result from a lack of repair mechanisms during replication
- Haploid
Plastid genes (i.e. Mitochondrial genes)
Differentiate type I and II markers
Type I - markers associated with genes of known function
Type II - associated with anonymous genomic segments
DNA Marker Technologies are dependent on:
*Gel electrophoresis
*Polymerase chain reaction (PCR) – for nucleic acids
*Separates nucleic acids or proteins on the basis of their rate of movement through a gel in an electrical field
*Makes use of either agarose or acrylamide gel matrix
Gel Electrophoresis
*Amplifies EXPONENTIALLY specific segments of DNA or RNA
*Pioneered by Kary Mullis
*Follows principle of DNA replication
Polymerase Chain Reaction (PCR)
3 Stages of PCR
Denaturation, Annealing, Extension
Stage wherein
- Makes the dsDNA single stranded
- Helicase absent; expose it to elevated temperatures to unwind
What is the temp?
Denaturation; 95 celsius
- Form H-bonds at specific sites where they are complimentary
- At 95 C no more H-bonds can form; lowering temperature to ___ in order for
free nucleotides to bind
Annealing; ~50 celsius
T or F: In Annealing, 72 C is the temperature where DNA polymerase optimally functions
F, In extension 72 C is the temperature where DNA polymerase optimally functions
- mRNAs found in cells can also be amplified
- Makes use of reverse transcriptase enzyme
- Produces ds copy DNA; From an RNA template, it produces a copy DNA
Reverse Transcriptase Polymerase Chain Reaction (RT PCR)
How to identify the mRNA and what is this trait’s function?
poly-A tail which protects the 3’ end of mRNA
Serves as primer binding site
Steps in RT PCR:
- Lyse cells and purify mRNA
- Hybridize with Poly(T) primer
- Make DNA copy with reverse transcriptase -> RNA-DNA complex
- Treat with alkali to degrade RNA -> makes ssDNA
- Hairpin loop will serve as a primer to amplify another round -> dsDNA
Name the following DNA tools and Techniques:
- RFLP
- RAPD
- AFLP
- SSCP
- DNA S
- Restriction Fragment Length Polymorphisms (RFLP)
- Randomly Amplified Polymorphic DNA (RAPD)
- Amplified Fragment Length Polymorphism (AFLP)
- Single-stranded Conformational Polymorphism (SSCP) Analysis
- DNA Sequencing
a. Look for variation in the sequence at the restriction site only
b. Type 1 marker - target is known
RFLP (Restriction Fragment Length Polymorphisms)
Makes use of radioactively labeled DNA probes
Developed by E.M. Southern
Not all fragments are visualized
Southern Blot Analaysis
Steps in Southern Blotting
- Restriction enzyme cuts the genome
- Run through electrophoresis
- Make a sandwich with a nitrocellulose filter, gel, sponge
- Transfer the bands to the nitrocellulose plate
- Submerge nitrocellulose filter containing probes
- Radio/fluoro enabled, shows band with probe attachment
Blotting targeting mRNA
Northern Blotting
Blotting targeting proteins
Western Blotting
Makes use of PCR
Type 2 marker - Target regions are unknown
Primers are short (10-20 nt)
RAPD (Randomly Amplified Polymorphic DNA)
Causes of variation in RAPDs
- Base substitutions at the primer binding sites
- INDEL between two RAPD primers
Type of marker used in AFLP (Amplified Fragment Length Polymorphism)
Type 2
Determines variations between DNA sequences
Makes use of ds PCR products
Type I marker used
PCR products are denatured and run on a gel matrix
Single-stranded conformational polymorphism (SSCP) analysis
Ultimate profiling method of variation
DNA sequencing
2 Methods of DNA sequencing
Maxam-Gilbert Method –enzyme cleavage
Sanger Method –chain termination
Steps in _________:
- Hydroshearing, Sonication, and enzymatic shearing
- Fragment Sequencing
- Fragment assembly of overlap
Shotgun Genome Sequencing
Enzymes with different amino acid
sequences but similar catalytic activity
Isozymes
Isozymes coded by different alleles of the same locus
Allozymes
DNA and RNA markers (4)
SNPs (Single Nucleotide Polymorphisms)
Indels (Insertions and Deletions)
STS (Sequence Tagged Sites)
VNTRs (Variable Number Tandem Repeats)
Most abundant type of genetic variation in humans
Different alleles exist in single base pair positions in genomic DNA
There is variability pair n the nucleotide present in the defined site in the DNA
SNPs (Single Nucleotide Polymorphisms)
Insertions or deletions of DNA in particular locations of a chromosome
Could be 10 bp to 1-5 kbp for transposable elements
Indel polymorphisms
Short DNA sequence that only occurs once and its location is known
Type I marker used
Rely on some degree of sequence knowledge
Sequence-Tagged Sites (STS)
Consists of multiple copies of tandemly arranged simple sequence repeats
(SSRs)
Mini and micro satellites
VNTRs (Variable Number Tandem Repeat)
11-16 bp repeated up to 1000 times
Scattered across the genome
Minisatellites
also known as short tandem
repeats (STR)
1-6 bp long repeated several times
for a total length of 100-200 bp
Microsatellites
