12/11/2013
DNA TOOLS AND TECHNIQUES
2. Gel Electrophoresis
•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
3. Restriction Fragment Length Polymorphisms (RFLP)
•Restriction enzymes – endonucleases
– cut DNA at specific sites
EcoRI – derived from E. coli
Restriction site
1
12/11/2013
Some examples:
•A single nucleotide change can make a difference
Wild-type allele
AGATCT
TCTAGA
Restriction site
Mutant allele
AGAGCT
TCTCGA
Not a restriction site
2
12/11/2013
Southern Blot Analysis for RFLP
•Makes use of radioactively labeled DNA probes
•Developed by E.M. Southern
•Not all fragments are visualized
3
12/11/2013
Southern Blotting
A D
B E
C F
•How to focus on specific regions of genome
Need a probe:
A short single stranded DNA which is
complementary to the region of interest
ATGGCATGGACC probe
GTCATATGTGTTCATGGCATGGACCGAGTCAATATGCGGCT
:::::::::::::::::::::::::::::::::::::::::
::::::::::::
CAGTATACACAAGTACCGTACCTGGCTCAGTTATACGCCGA
A probe will base pair to the region of interest
4
12/11/2013
NOTE:
Northern blotting – used on RNA
Western blotting – used on proteins
RFLP in Cystic Fibrosis
•Cystic fibrosis – autosomal recessive disorder
– accumulation of mucus in lungs
– affected individuals mutation in
CFTR protein
5
12/11/2013
RFLP analysis to detect VNTRs
•VNTR – variable number of tandem repeats
•VNTRs can be flanked by restriction sites
•Used in forensic DNA
VNTRs are classified as:
a. Minisatellites – 11-16 bp repeated up to 1000 times
– scattered across the genome
b. Microsatellites – also known as short tandem
repeats (STR)
– 1-6 bp long repeated several times
for a total length of 100-200 bp
6
12/11/2013
6
5
4
3
6
5
4
3
6 6
5 5
4 4
3 3
6,4 6,5 5,4 4,3 6,3 5,3
NOTE:
VNTRs can also be detected without using Southern Blots.
HOW?
6
5
4
3
6
5
4
3
7
12/11/2013
4. Randomly Amplified Polymorphic DNA (RAPD)
•Makes use of PCR
•Target regions are unknown
•Primers are short (10-20 nt)
Individual 1
1 2 3
DNA
4 5 6
PCR
Product A Product B
Individual 2
1 2 3
DNA
4 5 6
PCR
Product B
8
12/11/2013
RAPD profiles of 48 varieties of Korean sweet potatoes
9
12/11/2013
5. Single-stranded conformational polymorphism
(SSCP) analysis
•Determines variations between DNA sequences
•Makes use of ds PCR products
•PCR products denatured and run on a gel matrix
5. Single-stranded conformational polymorphism
(SSCP) analysis
WILD MUTANT
-
+
Gel matrix
10
12/11/2013
Genetic variation in global populations of Achatina
fulica (Bowdich, 1822) based on the 16S rRNA gene
I.K. C.Fontanilla1,2, C. Hudelot1, F. Naggs3, and C.M. Wade1
1Institute of Genetics, University of Nottingham, Queen’s Medical Centre, Clifton Boulevard, Nottingham NG7 2UH, UK
2Institute of Biology, College of Science, University of the Philippines, Diliman 1101, Quezon City, Philippines
3Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK
Gel profiles of the 15 haplotypes detected Nucleotide variations among the
from PCR-amplified 310-bp fragment of 16S haplotypes and their positions
rDNA gene in global populations of the giant
African land snail Achatina fulica
Nucleotide Position
1 1 11 1 1 1 1 2 2 2 2 2 2 2
1 4 0 0 45 5 5 5 9 0 1 5 8 8 8 8
9 2 2 6 51 5 6 8 7 5 7 8 1 2 5 6
. . . . .. . . . . . . . . . .
Haplotype A GACCCATAATTAAT TTT
Haplotype B AG............ ...
Haplotype C A.......G..... ..-
Haplotype D A............. ..-
Haplotype E AC......G..... ..-
Haplotype F A.....C.G..... ..-
Haplotype G A..T.......... ..-
Haplotype H A.......G..... ...
Haplotype I A.T..G.T.C.... ...
Haplotype J A.T..G.T...... A.-
Haplotype K A...........G. .--
Haplotype L A.G..G.T.CC... .--
Haplotype M A.T..G.T.C.... ..-
Haplotype N A.T.TG.T...G.. ..-
Haplotype O A...T........A..-
11
12/11/2013
P India Nepal Burma Thailand
67%
C Florida
33% E
8%
C
C
100%
C
100%
Philippines C
100% 100%
C Hawaii
92%
C
100%
Seychelles
O C
100%
Martinique
100%
R Uganda C
C
M N 100%
100%
3%
5% 5% I
F
27% Ogasawara 100%
C
L 100% Q
25% J
5%
5%
Barbados
F
H Sri Lanka 100% D
Tanzania K D 4%
26%
30% H A 20%
B C
2% 2% D C
G 2% 69%
C 50% 50%
2% C C C
12% C
C C 100%
100% 100% 100%
76% 100% New Caledonia Ecuador
D
80%
Mauritius Penang (Malaysia) Singapore Sabah (Malaysia) French Polynesia Bolivia Brazil
Mayotte
Distribution map of the Achatina (Lissachitna) fulica populations and their 16S rRNA haplotypes.
6. DNA Sequencing
•Two methods (developed in the late 1970s):
a. Maxam-Gilbert Method – enzyme cleavage
b. Sanger Method – chain termination
12
12/11/2013
Maxam-Gilbert Method
Sanger Method
13
12/11/2013
Sanger Method
14
12/11/2013
PCR-Direct Sequencing using the Sanger Method
ABI 3130xl Genetic Analyzer
15
12/11/2013
>AU_Cv_EST_015A_F05.ab1
NNNNNNNNNNTNNNNNNNNNNNNCNCANCCGANCGANCGANCNNAGCGNGNNNNGAGCGAGGAAGCGGCCGCNNNNNNNNNNNNNNN
TNNNNAANNNNNANNNNNGGNCCGGANNNNNCCCGGGANNNNGCCNTTNNGCCGGGGGATTTGGNNNGTNGAAAAACAACATGGAGAAA
GTTGTGANTNNNTTGCCGAAGNTNNNNTTTAGTTTACGGACACATTTTTGNNTTGGAACTAAACAACGGAATAGACTTGGATTATTTGTGGATTGC
GAATGCCAAAATTTACAGTGATTCAGTCGGTGAGGNCCCTGTTTTGTGAGTTTGGATTATTAACGAAGTACGTACTGCACACATTGCTTTGTTGAACG
ATGCTAATTGTTGTGGTTTTTCCGGTGAACGATGGGCATCAAATACACAGGCAATACAAATTGAATGTGTAGATTTGTTTGTTTGAGAAAATCTTATTCT
AGCTNAGAGATTAGACATGGTAACAATCAATTCGTTTTGCCTCGATGATGGCGTCCGGAAAATTTTACTTATGTGAGGTGATGAGAGTATGACCGTAT
AAACCAGAAAGGAAACAGNCCAGCGCCCATTTTGNCATCACNGAAAAAATCACCANGAGAGAAATNGTNCATCTTCAGACAGGNCAANGNGGAA
ACCAAATCGGTNNNACGTNCNGGGAAGNAANNNCGGATGAGCACGGNNNCGACCCCACNGGAACCNNNNANGGNGACNCAGACCTTCAGCN
NGAGAGAANCAANGNNNNCNACAANNAANCAACNGNN
16
12/11/2013
DNA Marker Technologies
•Paved the way for genomics
•Evaluate genetic variation within and among individuals,
species, and higher order taxonomic groups
All organisms are subject to mutations as a result of
normal cellular operations or interactions with the
environment, leading to genetic variation (polymorphism).
DNA Marker Technologies
• For genetic variation to be useful to geneticists, they
must be:
1. heritable
2. discernible to the researcher, whether as a
recognizable phenotypic variation or as a genetic
mutation distinguishable through molecular
techniques
17
12/11/2013
DNA Marker Technologies
• Genomic sources:
• Nuclear genes
• Usually come in pairs in diploid organisms
• May occur as different alleles in heterozygotes
• Mitochondrial genes
• Haploid
• Maternally inherited
• More variable than nuclear genes due to rapid mutation
• result from a lack of repair mechanisms during
replication
DNA Marker Technologies
Types of Markers
Type I
• markers associated with genes of known function
Type II
• associated with anonymous genomic segments
18
12/11/2013
Molecular Markers for Molecular Tools & Techniques
•Protein markers
•Isozymes
•DNA and RNA markers
•SNPs
•Indels
•STS
•VNTRs
A. Protein markers
1. Isozyme Analysis
•Isozymes – enzymes with different amino acid
sequences but similar catalytic activity
•Allozymes – isozymes coded by different alleles of
the same locus
•Used in population genetic studies for low-level genetic
variations
•Some examples:
•Alcohol dehydrogenase (ADH)
•Malic dehydrogenase (MDH)
•Acid phosphatase (ACP)
19
12/11/2013
NOTE:
20
12/11/2013
Molecular Markers for Molecular Tools & Techniques
•Protein markers
•Isozymes
•DNA and RNA markers
•SNPs
•Indels
•STS
•VNTRs
B. DNA & RNA markers
1. Single Nucleotide Polymorphisms (SNPs)
• Single base pair positions in genomic DNA at which different
sequence alternatives (alleles) exist in normal individuals in
some population(s)
• individuals in the population may differ in identity of the
nucleotide pair present at a particular defined site in the
DNA
• most abundant type of genetic variation in humans
21
12/11/2013
B. DNA & RNA markers
1. Single Nucleotide Polymorphisms (SNPs)
GATTTAGATCGCGATAGAG
GATTTAGATCTCGATAGAG
^
B. DNA & RNA markers
1. Single Nucleotide Polymorphisms (SNPs)
RECALL:
PURINES PYRIMIDINES
A C
TRANSITION TRANSITION
G T
TRANSVERSION
22
12/11/2013
RECALL: SSCP analysis of Achatina fulica populations
Gel profiles of the 15 haplotypes detected from PCR- Nucleotide variations among the haplotypes and
amplified 310-bp fragment of 16S rDNA gene in global their positions
populations of the giant African land snail Achatina fulica
Nucleotide Position
1010 1415 1515 15 19202125 2828 2828
19 422 6 5 1 5 6 8 7 5 7 8 1 2 5 6
. . . . . . . . . . . . . . . .
Haplotype A GAC CC AT AAT T AAT T T T
Haplotype B AG. . . . . . . . . . . . . . .
Haplotype C A. . . . . . . G. . . . . . . -
Haplotype D A. . . . . . . . . . . . . . . -
Haplotype E AC . . . . . . G. . . . . . . -
Haplotype F A. . . . . C . G. . . . . . . -
Haplotype G A. . T . . . . . . . . . . . . -
Haplotype H A. . . . . . . G. . . . . . . .
Haplotype I A. T . . G. T . C . . . . . . .
Haplotype J A. T . . G. T . . . . . . A. -
Haplotype K A. . . . . . . . . . . G. . - -
Haplotype L A. G. . G. T . C C . . . . - -
Haplotype M A. T . . G. T . C . . . . . . -
Haplotype N A. T . T G. T . . . G. . . . -
Haplotype O A. . . T . . . . . . . . A. . -
B. DNA & RNA markers
2. Insertion/Deletion polymorphisms (Indels)
• insertions or deletions of the DNA at particular
locations on the chromosome
• could be fewer than 10 bp or 1-5 kb for transposable
elements
23
12/11/2013
B. DNA & RNA markers
3. Sequence-tagged sites (STS)
• a short DNA segment that occurs only once in
the human genome and whose exact location
and order of bases are known
• unlike arbitrary primers, STS rely on some degree
of sequence knowledge
•E.g. markers used to detect disease-causing alleles by
binding them to radioactively labeled probes
Recall: RFLP in cystic fibrosis
•Cystic fibrosis – autosomal recessive disorder
– accumulation of mucus in lungs
– affected individuals mutation in
CFTR protein
24
12/11/2013
B. DNA & RNA markers
4. Variable number of tandem repeats (VNTRs)
• consist of multiple copies of tandemly arranged simple
sequence repeats (SSRs)
• Recall the different types of VNTRs:
a. Minisatellites – 11-16 bp repeated up to 1000 times
– scattered across the genome
b. Microsatellites – also known as short tandem
repeats (STR)
– 1-6 bp long repeated several times
for a total length of 100-200 bp
Recall: VNTR analysis
6
5
4
3
6
5
4
3
6 6
5 5
4 4
3 3
6,4 6,5 5,4 4,3 6,3 5,3
25
