Molecular Techniques Flashcards
restriction enzymes
cleave palindromic sequences with a double stranded cut; made by bacteria
unexpected pattern of digestion
indicates polymorphism - deletion or substitution, insertion, methylation change
agarose gel electrophoresis
separates DNA based on size; longer DNA will move shorter distances
separated DNA segments used for _
cloning, generating a probe, purifying a desired segment for DNA sequence determination
capillary electrophoresis
electrophoretic DNA separation without a gel
DNA cloning
ability to generate desired DNA fragments of DNA
DNA cloning steps
restriction enzymes cleave restriction site to produce sticky sites –> foreign DNA with gene of interest contains matching sticky ends so it will hybridize –> recombinant vector carrying gene of interest is placed in presence of antibiotics –> only recombinant vectors will grow and divide because of antibiotic resistance gene
plasmid components
OriC, restriction site, and antibiotic resistance gene
What is useful for cloning large segments of DNA?
artificial chromosomes
What is useful for cloning shorter sequences of DNA?
PCR and recombination methods
DNA library
cloned segments from entire DNA or RNA (cDNA) complement of target of interest carried in a vector
libraries can be made from _
genomic DNA, cDNA from total mRNA, or subset of either
DNA hybridization (heteroduplex)
ssDNA is annealed to complementary single stranded sequence
Southern Blot
restriction enzymes digest genomic DNA and fragments are separated in agarose gel –> sequences are transferred to Southern blot for hybridization
Southern blot is used for _
specific detection of specific sequences (ex. sickle cell)
Western blot
proteins
Northern blot
RNA
selective hybridization
can be used with small segments of DNA to detect single mismatches
How does selective hybridization work?
if the DNA segment of interest will not anneal to the known complementary DNA, it is because there is a mismatch which indicates disease
PCR steps
denature, anneal to primer, extending stage
PCR requirements
thermostable polymerase, single stranded primer, dNTPs and needed enzymes
PCR can also be _
quantitative; determine how much was in original sample
real-time quantitative PCR
the more you start with, the less cycles you will need to get to a detection point (exponential phase)
limitations of PCR
must be able to define endpoints with two primers, any error will be amplified, GC sequences difficult to amplify, polymerases eventually fail
dideoxy nucleotides
both 2’ and 3’ C’s lack hydroxyl groups, terminating the chain when they bind
Sanger dideoxy sequencing
four tubes with low concentrations of radio-labeled ddNTPs are mixed with regular dNTPs and DNA polymerases –> DNA will elongate until reaches a ddNTP –> eventually results in multiple lengths with a known end which can tell you the sequence of the DNA
limitations of Sanger sequencing
sequence obtained is of newly synthesized strand, rather than template, so errors possible, polymerases don’t last forever, not large enough for genome-scale analysis
exome
complete exon complement
detection of large DNA segments
chromosome banding karyotype (>100kb)
detection of specific large DNA segments
southern blot hybridization or FISH (>10kb)
balanced translocation
all information is there but in a different spot
differential detection of normal vs. altered sequences or sequence copy number
selective or allele-specific hybridization, CGH
amplification of defined or less defined set(s) of sequences
CGH, PCR, DNA sequencing
unbalanced translocation
missing material
isochromosomes are examples of _
a balanced translocation
paracentric inversion
rearrangement on one side of centromere (within p or q arm)
pericentric inversion
rearrangement around the centromere (p to q or vice versa)
FISH and karyotyping require _
metaphase cells because the chromosomes are condensed
When can FISH be done with interphase cells?
if looking for duplication or loss
comparative genome hybridization
used to detect copy number changes only
karyotyping uses _
detecting suspected or unknown aneuploidy and large structural rearrangements (deletions, inversions, translocations)
karyotype disadvantages
difficulty in automation, must use metaphase cells (takes a while to induce)
chromosome paint
FISH that covers the whole chromosome in certain color (probe sequences must be unique so color is not repeated on different chromosome)
RFLP
used to identify suspected changes (gene level) –> based on Southern blot hybridization
comparative genome hybridization (CGH)
can be used when you have no idea what’s wrong but will only detect copy number variations
CGH method
sample DNA and reference DNA are hybridized –> normal genes will hybridize completely and form mixed color
too much sample DNA in CGH
indicated a duplication
too much reference DNA in CGH
indicates deletion
SNPs
single nucleotide polymorphisms
methods to detect SNPs
exome sequencing, whole genome sequencing, targeted enrichment for sequencing, gene-specific microarray
micro-array based mutation scanning
will show the nucleotide in a normal sequence and then will show the nucleotide in diseases sequence
targeted gene sequencing
sample DNA is cut into fragments and allowed to hybridize with reference DNA –> targeted sequences are eluded and amplified –> determine sequence
advantages of targeted gene sequencing
efficient for scanning many segments or long genome regions, detects all smaller variant types, and less expensive than more global sequencing techniques
whole exome sequencing
all annotated exons are targeted
methods to detect changes in cytosine methylation
sodium bisulfite treatment followed by PCR and DNA sequencing of products
sodium bisulfite
will cause unmethylated cytosines to deaminate into uracils –> will eventually have T’s in PCR analysis
