Lecture 14 Flashcards
A genomic library is
a collection of clones from one species’ genome
ex. DNA is cut into pieces (fragments) by restriction enzymes and fragments are inserted into vector. (vector has 1. selectable marker 2. origin of replication 3. polylinker)
cDNA is
complementary DNA synthesized from mRNA
DNA →
RNA →
mRNA (starting point for creating cDNA library) has polyA tail→
single strand cDNA →
double strand cDNA
cDNA libraries reflect
gene expression (transcription)
a DNA library can be
1) genomic 2) cDNA
Making a cDNA library
1) prepare poly(A) + RNA from desired source
2) synthesize cDNAs
3) ligate with vector (plasmid or ɣ)
steps to synthesize cDNAs (making cDNA library)
1) add poly-A tail to mRNA
2) anneal primer (oligo dT)
3) synthesize cDNA with reverse transcriptase
4) degrade mRNA with NaOH
5) synthesize second cDNA with DNA pol 1
6) nick loop with S1 nuclease
7) Ligate with vector
cDNA vs genomic libraries
cDNA - no introns or non-translated regions (only whats expressed)
genomic libraries - represent all sequences in the entire genome regardless of function
cDNA libraries contain:
- only exons present in mature mRNAs, - not introns, 5’ or 3’ regulatory sequences
- only the genes that are transcribed in the cells/tissues/developmental stages interrogated
- potentially drastically different numbers of clones reflecting strong differences in expression level among genes
Would genomic DNA libraries be the same between two different tissues from same individual
yes, same
if cDNA → No
Molecular probes detect genes or gene products of interestthrough
complementary base-pairing or antibody-protein binding (affinity)
molecular probes → detect specific sequences
Principle of molecular hybridization
Exploits ability of ____
can be done ___
single-stranded DNA to base-pair with complementary sequence, or ability of antibodies to bind proteins
Can be done in solution, or on a solid support
Hybridization of labeled probe is used to
(techniques with molecular hybridization)
- identify clones in libraries (genomic, cDNA)
- identify bands in gels (southern, northern, western)
- identify chromosomes, chromosomal locations (fluorescent in situ hybridization = FISH)
- identify tissues where a gene or protein are expressed
- many other applications (e.g., microarrays…)
Identifying clones in libraries (using labeled probes)
bacterial colonies are grown
each colony → piece of genomic DNA
all colonies → all DNA in library
Transfer colonies to nitrocellulose filter
On filter → cells are lysed and DNA is denatured radioactively labeled nucleic acid probe is added
some radioactively labeled nucleic acid probes are hybridized to DNA of some colonies
exposed to x ray → complementary to probe → lights up
Identifying bands in gels (using labeled probes)
step 1 → electrophoresis of DNA/RNA/protein through (agarose) matrix
step 2 → blotting to membrane
step 3 → following hybridization with labeled probe, filter exposed to X-ray film or analogous detection system
spots on the X-ray film correspond to DNA bands in gel
Southern Blot
DNA
Northern Blot
RNA
Western Blot
protein
Identifying chromosomes or chr. regions (using labeled probes)
FISH: Fluorescent in situ hybridization
DNA probe → complementary to specific region on chromosome
Application: revealing abnormalities in chromosome number (e.g., trisomy 21)
used to determine chromosomal location of a gene
used to dertermine distrabution of specific mRNA
Identifying expression patterns in tissues or whole organisms (using labeled probes)
blue → where gene is expressed
shows v specific expression pattern
Drosophila early embryos:
blue stripes (both panels) are eve gene expression, red (bottom panel) is Kruppel expression, and each green dot represents a single nucleus.
Microarray hybridizations (using labeled probes)
Microarray → a collection of probes attached to a solid support
used to assess transcriptional patterns (mapping transcribed regions comparing levels of gene expression)
and mapping DNA divergence between genomes
yellow - equal expression in both
types of sequencing
Sanger (dideoxy) sequencing - 1970
Massively parallel sequencing →
Next-generation sequencing (NGS) or 2nd generation sequencing
→high accuracy but sequencing reads1 are short
Single molecule sequencing (long read) → 3rd-generation sequencing
→ error-prone but sequencing reads are long
sequencing “read”
bases sequenced from an individual sequencing reaction
Sanger sequencing steps
poly starts elongating → any time has potential encounter specific ddTTP (here A)
uses dideoxynucleotides (ddNTPs) →chain terminators → once incorporated into growing chain next not added
run through gel
dideoxynucleotides (ddNTPs)
are missing both (2) hydroxyl groups (3’)
are chain terminators because missing hydroxyl group nessary for addition (has group for phosphate)
Analyzing Sanger sequencing gell
need 4 rxns → for 4 nucleotides
smallest → bottom so read bottom to top to find sequence (after primer sequence)
When sequencing in the presence of dG, dC, dT and ddA (no dA present), the longest possible molecule seen on a
sequencing gel is:
12 nts (because stops at 1st A, b/c no dA only ddA)
What is the sequence of the newly synthesized strand?
5’-C T A A C G
Automation of Sanger sequencing
increased efficiency
Slab gels with radioactive dNTPs
Capillary sequencing with 4 fluorescently labeled dNTPs
96 samples per run
30-60 kb per run
HGP used this method
Next generation sequencing steps
chop DNA up (ex. sonificatoin)
randomly sheered sequence → is sequenced → use bioinformatics to put it all back together
Illumina sequencing
next gen sequncing
terminator is reversible
third generation sequencing
SMART - single molecule real time sequencing
allow much longer fragments to be sequenced (produces longer reads)
PacBio sequencing
Nanopore sequencing
NGS Improvements
+ downside
Speed (massively parallel sequencing)
Low cost (miniaturization)
Low amount of template DNA (no cell-based cloning)
Accuracy through redundancy
downside is smaller read length
150 nts
Application of NGS
portable real-time sequencing system used in Guinea during 2015 Ebola epidemic
data generated in 24h of receiving viral sample
epidemiological tracking, identify virulence mutations, inform therapy
You know that a gene has one intron.
A colleague has cloned this gene and given it to you to study, but forgot to tell you if clone is cDNA or gDNA.
How can you use PCR to figure this out? (Assume you know the complete genomic and cDNA gene sequence)
The most effective way to answer this without ambiguity is to design PCR primers that flank the intron (annealing in the bordering exonic regions).
Either way, whether the clone were cDNA or genomic DNA, an amplicon would result.
However, if it were a genomic clone containing the intron, the amplicon size would be predictably larger than if it were a cDNA
Ans → B
A → cDNA → only complement to mRNA (no intron)
C → antibody → protein
D → promoter → not in cDNA
Ans → A
B → not know sequence, not have specific probe
C → not know primer
D → same problem
E → for proteins
Ans → B
A → has intron and regulatory sequence → not work (want protein → need DNA seq corresponding to protein)
D
D
D
B
C
C
D
shotgun cloning
clone first search later -
genes located by creating libraries of DNA sequences then searching libraries for genes of intrest
positional cloning
isolation of genes on the basis of their position on a gene map
use maping studies to determine linkage between molecular makers and phenotype
use chromosome walking - obtain new fragment, continue overlapping until ding dene of intrest
DNA fingerprinting
or DNA profiling
some parts of the genome are highly variable, each person’s DNA sequence is unique and, like a traditional fingerprint, provides a distinctive characteristic that allows identification.
Most DNA fingerprinting uses microsatellites, or short tandem repeats (STRs), - Very short DNA sequence repeated in tandem
