Unit 2
chain-terminator sequencing
(Sanger sequencing) enzymatic synthesis of polynucleotide chains complementary to "template" DNA; synthesized chains are terminated at specific nucleotide positions; resolved the fragments on a polyacrylamide gel to obtain the sequence.
microsatellites?
(or simple tandem repeats (STRs)) • Di-, tri- or tetra-nucleotide repeat units • Randomly distributed in genomes • Shorter than VNTRs (typically 10-30 repeat units) • Generated by "slippage" during DNA replication
differences between agarose gel electrophoresis and polyacrylamide gel electrophoresis
- - - -
YACs to make clones of DNA?
- A commercial ready-to-use circular bacterial plasmid containing ori, 1 restriction site, 2 selectable markers, 1 centromere, and 2 Telomeres. - Use BamHI to digest the plasmid, producing a linear chromosome with two telomeric ends - Use restriction enzyme to cleave the linear chromosome to produce two arms, each with a selectable marker. - Use Ligase to ligate the fragment of genomic DNA into the linear chromosome (YAC) - Transform the YAC into yeast spheroplast and use positive selections to select yeast cells with YAC clone that both markers are expressed
what are the factors that affect Tm? (factors affect hybrid stability) -
- Base composition ((G+C)/(T+A) ratio): Tm increases with increased G+C content - Length of nucleic acid sequence - Degree of sequence similarity between hybrids: every 1% mismatching of bases in a DNA duplex reduces the Tm by 1°C. - Temperature: the reaction temperature affects the rate of nucleic acid hybridization. - Other factors: ionic strength, denaturing agents, etc.
what are the three steps in PCR?
- Denaturation denature the target sequence (template) into single-stranded DNA (90-94 °C) - Annealing anneal the oligonucleotide primers to the single-stranded template DNA (depends on primers, but typically 45-60 °C) - Extension synthesize complementary strands (depends on polymerase: Taq polymerase, 72 °C)
procedure of Northern blotting?
- Purification of DNA/RNA: Extract and purify the RNA from either cells or tissue sources. - Gel electrophoresis: Separate RNA samples on agarose gel with formaldehyde as the denaturing agent that limits secondary structures of RNA molecules. - Transfer: Transfer the DNA/RNA fragments from the gel onto a nylon membrane. - Prehybridization (Blocking): Wash the nylon membrane with a prehybridization solution to block non-specific DNA interactions and reduce background noise. - Preparation of probe: Prepare fresh probe DNA and label with 32P alpha-labeled dCTP. - Hybridization: Incubate the blot with labeled probe. - Detection of probe
How to detect labelled molecules? -
- Radioactively labelled molecules can be detected with X-ray sensitive film ( autoradiography) or a radiation-sensitive phosphorescent screen (phosphorimaging) - Non-radioactive: *molecules labeled with fluorophores (dyes) with defferent emission wavelengths can be detected with film or fluorescence detector *molecules labeled with chemiluminescence can go through chemical reactions to generate light signals detected with film
Southern blotting:
- a method used in molecular biology for detection of a specific DNA sequence in DNA samples. It combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.
what are needed in chain-terminator sequencing?
- single-stranded template DNA; - oligonucleotide primer; - polymerase; - all four dNTPs (large amounts), one can be radioactive labeled; - one of four ddNTPs, at very low concentration. [in each well of the gel, it contains fragments that are made from all four dNTP and one ddNTP, for example, dATP, dCTP, dTTP, dGTP, and ddATP)
What are BACs?
-Bacterial artificial chromosomes -Are simply plasmids designed to propagate large DNA fragments -propagated at low copy number in E. coli -100-300+ Kb fragments can be maintained
What are differences between genomic library and cDNA library?
-Genomic library contains fragments of genomic DNA (genes): partial digestion allows production of overlapping fragments, used to construct as contiguous chromosomal regions and therefore the DNA sequences; contain promoters and introns; independent to gene activity; cannot express in heterologous system; useful for genome analysis, map-based cloning, promoter studies, etc. -cDNA library contains DNA copies of cellular mRNAs: reverse transcriptase makes a DNA copy of an RNA; add RNaseH (specific for the RNA strand of an RNA-DNA hybrid) and carry out a partial digestion, short RNA segments are served as primers for DNA synthesis; do not contain promoters and introns; dependent to gene activity (reflects gene activity); can express in heterologous system; useful for analysis of coding regions and gene functions
"Shotgun" sequencing a genome using Sanger technique
-Randomly sheared DNA used to make a genomic library -Clones picked at random from the library and sequenced using universal primers -Individual sequence reads assembled in computer to produce contigs -Contigs put together to produce a genome 'assembly'
What are three ways to ligate the DNA fragments with plasmid to produce vectors?
-Use type II restriction enzyme to produce two sticky ends and then anneal and ligate the DNA fragment with plasmid together - TA cloning: - TOPO-TA cloning
What are YACs?
-Yeast artificial chromosomes -Linear DNA fragments that can be replicated in yeast -Contain yeast replication origin (ori), centromere (CEN), telomeres (TEL) Used to clone fragments up to 2,000 Kb
What is cloning?
-isolation of a particular nucleotide DNA sequence from its genomic context -production of multiple identical copies of that sequence
Melting temperature
-the temperature at which half of DNA double helices dissociating into single strands. (The temperature at which half of the nucleic acid strands are denatured) - usually experimentally determined by measurement of absorbance at 260 nm
what are two types of primers used in DNA sequencing?
-universal primer -internal primer
What are three essential features of plasmid vectors?
1. Origin of replication (ori site) 2. Selectable markers—cells carrying the plasmid need to be distinguished from those that lack the plasmid 3. Unique restriction enzyme cleavage sites for the insertion of DNA sequences that are to be cloned, most are located in a 'polylinker' or multiple cloning site (MCS)
TOPO-TA cloning
3'-A tailed double-stranded DNA fragment; TOPO vectors with topoisomerase I covalently bound to vector(T-P-topoisomerase at 3' end) the 5'-OH group of the DNA fragment can attack the TOPO vector and release the topoisomerase I enzyme, produce a ligated circular vector
Northern blotting:
A technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample.
microarrays:
DNA chip or biochip; a collection of microscopic DNA spots attached to a solid surface. Used to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome
what is directional cloning? How is it produced?
Digesting a DNA insert or vector molecule with two restriction endonuclease enzymes to create a blunt end and a sticky ends at each restriction fragment. By introducing asymmetry into the system, it can make sure the direction of the DNA fragment in vector is unique. (important when studying expression of genes)
T/F? Chain terminator sequencing needs equal amount of all four dNTPS and one radioactive laveled ddNTP.
F. it need large amount of all four dNTP, and one need to be labled, and small amount of unlabeled one of four ddNTP.
what are forces that affect nucleic acid stability? -
Forces that tend to separate helix into random coils: - electrostatic repulsion of side chains (negative charges on phosphate group) - higher entropy state of random coil ( more disorder, so more favourable) Forces that tend to stabilize helix: - hydrogen bonding between complementary base pairs; - base stacking force (van der Waals interactions between aromatic rings of base pairs)
Tm : -
Melting temperature
What are two types of SSLP?
Minisatellites (variable number of tandem repeats—VNTRs) Microsatellites (or simple tandem repeats (STRs))
what is PFGE
PFGE is necessary because during "continuous field" gel electrophoresis, DNA fragments above 30-50 Kb migrates with the same mobility regardless of size. • With PFGE, DNA fragments are forced to change direction when the electrical field is re-oriented • With each re-orientation, smaller sized DNA fragments begin to move in the new direction more quickly than the larger ones, allowing separation to occur
PCR
Polymerase chain reaction: a technology in molecular biology used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
why is PFGE useful than old gel electrophoresis technique?
Pulsed-field gel electrophoresis (PFGE) is a highly discriminative molecular typing technique that is used in epidemiological studies worldwide. PFGE is based upon the variable migration of large DNA restriction fragments in an electrical field of alternating polarity
How to synthesize and label nucleic acid probes? -
Random Primers DNA Labeling System: a method to radioactively label DNA; 1. denature DNA fragments by heating in boiling water 2 Anneal random sequence oligonucleotides to both strands. Klenow fragment polymerase is then used to extend the oligonucleotides, using three cold nucleotides and one radioactively labeled nucleotide provided in the reaction mixture, to produce a uniformly labeled double-stranded probe. Each batch of random oligonucleotides contains all possible sequences (for hexamers, which are most commonly employed, this would be 4096 different oligonucleotides) and so any DNA template can be used .)
Shotgun sequencing:
Randomly breaking up DNA sequences into lots of small pieces and then reassembling the sequence by looking for regions of overlap. DNA is broken up randomly into numerous small segments, which are sequenced using the chain termination method to obtain reads. Multiple overlapping reads for the target DNA are obtained by performing several rounds of this fragmentation and sequencing. Computer programs then use the overlapping ends of different reads to assemble them into a continuous sequence.
Where do restriction enzymes come from?
Restriction enzymes are found in bacteria. Bacteria use restriction enzymes to kill viruses - the enzymes attack the viral DNA and break it into useless fragments.
RFLP?
Restriction fragment length polymorphisms When restriction fragment lengths vary due to the presence / absence of restriction sites from sample to sample (e.g., variation within human populations) Brown 1999. Genomes 5
difference between RFLP & SSLP?
SSLPs can be multiallelic
SSLP?
Simple sequence length polymorphisms Arrays of repeat sequences that display length variations, with different alleles containing different numbers of repeat units. • Unlike RFLPs, SSLPs can be multiallelic: each SSLP can have a number of different length variants
procedure of Southern blotting:
Step 1: DNA digestion Digest DNA with a restriction enzyme Step 2: Gel electrophoresis Separate DNA by gel electrophoresis, usually an agarose gel. Denature DNA into single strands by incubation with NaOH. Step 3: Blotting Transfer DNA to a positively charged nylon membrane. The DNA fragments retain the same pattern of separation they had on the gel. Step 4: Probe labeling Step 5: Hybridization & washing Incubated the blot with DNA probe which is single-stranded to form hybridization to its complementary DNA sequence. After hybridization, the unhybridized probe is removed by washing in several changes of buffer. Step 6: Detection The bound, labeled probe is detected using the method required for the particular label used. For example, radiolabeled probes may be detected using X-ray film or a phosphorimaging instrument, and enzymatically labeled probes are typicallly detected by incubating with a chemiluminescent substrate and exposing the blot to X-ray film.
Renaturation / annealing
The process by which complementary strands of nucleic acids re-form their native conformations. (The joining together by typical base pairing of two fully separated complementary sequences, resulting in native duplex structure.
Renaturation / annealing: -
The process by which complementary strands of nucleic acids re-form their native conformations. (The joining together by typical base pairing of two fully separated complementary sequences, resulting in native duplex structure.
Hybridization: -
The process of forming a double helix from two complimentary single strands of nucleic acid
T-tailed vector
The target vector is linearized and cut with a blunt-end restriction enzyme. The vector is then tailed with dideoxythymidine triphosphate (ddTTP) using terminal transferase to ensure the addition of only one T residue. This tailing leaves the vector with a single 3'-overhanging thymine residue on each blunt end. It can be ligated to "A-tailed" PCR product
why we choose type II restriction enzymes in recombination DNA or cloning?
They form homodimers, with recognition sites that are usually undivided and palindromic and 4-8 nucleotides in length. They recognize and cleave DNA at the same site, and they do not use ATP or AdoMet for their activity—they usually require only Mg2+ as a cofactor (easy to control termination).
Why type I restriction enzyme is not useful?
Type I enzymes: complex, multisubunit, combination restriction-and-modification enzymes that cut DNA at random far from their recognition sequences. of considerable biochemical interest, but they have little practical value since they do not produce discrete restriction fragments or distinct gel-banding patterns.
Which type of restriction enzymes we use in recombination DNA or cloning?
Type II
isoschizomers
Type II restriction enzymes that recognize the same DNA sequence; they may or may not cleave at the same position.
TA cloning
Use DNA polymerases to add a single 3'-A residue to double-stranded DNA fragment via a terminal transferase-like activity; the A-tailed DNA product is ligated with a linearzed vector with 3'-T overhangs by DNA ligase. (PCR product with a single template-independent base addition of an adenine (A) residue to the 3' end of the PCR product, through the normal action of the polymerase. These "A-tailed" products are then ligated to a complementary T-tailed vector using T4 DNA ligase, followed by transformation)
procedure in automated DNA sequencing
Uses fluorescently-labeled ddNTPs; • A PCR-like reaction (with only one primer) is used to incorporate the dNTPs and ddNTPs and produce labeled chain-terminated products • All four synthesis / chain termination reactions done in the same tube on a thermal cycler • "Capillary" gel electrophoresis is typically used to separate DNA fragments (faster than 'standard' gels, allows more samples to be processed in parallel) • Sample run in a single lane on the gel, runs past a laser beam which causes fluorescence • Nucleotide-specific fluorescence received by a detector, converted into a "chromatogram"
contig
a contiguous set of overlapping DNA sequences In bottom-up sequencing projects, a contig refers to overlapping sequence data (reads); in top-down sequencing projects, contig refers to the overlapping clones that form a physical map of the genome that is used to guide sequencing and assembly.
what is chromosome walking?
a method of positional cloning used to find, isolate, and clone a particular allele in a gene library. Starting with the DNA sequence of a single clone, probes can be used to "screen" a genomic library for additional clones that contain fragments overlapping with the original clone. Repeat as necessary.
electrophoresis
a technique used in laboratories in order to separate macromolecules based on size. The technique applies a negative charge so proteins move towards a positive charge. This is used for both DNA and RNA analysis.
How to isolate intact chromosomes
cells are embedded in low-melt agarose and digested with a protease (e.g., proteinase K) to remove proteins, in particular, those attached to the DNA (e.g., histones)
eukaryotic features of YACs
centromere (CEN), telomeres (TEL)
restriction endonucleases
enzymes that catalyze the double -strand cleavage of DNA at specific or non-specific sequences
type of primers
primers used for DNA sequencing depends on: • The type of vector • The size of the insert
procedure of microarrays: -
principle: hybridization between two DNA strands;
what is qPCR?
quantitative PCR or 'real-time' quantitative PCR: • Used to quantify the abundance of a particular DNA sequence in a sample. • Uses fluorescence to measure amplification of target sequence as it happens, cycle by cycle (the rate of PCR amplification is related to target abundance in the sample being used as template) • Often combined with RT-PCR to study differences in gene expression from sample to sample.
what is RT-PCR?
reverse transcriptase PCR: • Synthesize cDNA from RNA using "reverse transcriptase" • Use cDNA as "template" for PCR (see cDNA synthesis)
STR?
simple tandem repeats (ormicrosatellites)
Denaturation / melting
the process of separation of double-stranded DNA into single strands. (The process of separating the polynucleotide strands of duplex nucleic acid.
How many different types of restriction enzymes?
three
VNTP
variable number of tandem repeats (minisatellites)
What are required to carry out PCR?
• A thermostable DNA polymerase (usually Taq polymerase) • A pair of oligonucleotides ("primers") • A small amount of "target" DNA (template) • dATP, dCTP, dGTP, dTTP • A repeated series of temperature changes to allow polymerase-catalyzed DNA synthesis reactions happening
Parameters to vary in PFGE
• Agarose concentration • Field strength (volts / cm) • Run time (hours) • Pulse time (seconds / minutes) • Re-orientation angle (usually 120°)
steps in chain-terminator sequencing
• Also called dideoxy sequencing or Sanger sequencing • Enzymatic synthesis of polynucleotide chains complementary to "template" DNA • Synthesized chains are terminated at specific nucleotide positions • Synthesized fragments resolved on a polyacrylamide gel. Steps: 1. Preparation of single-stranded "template" DNA 2. Anneal oligonucleotide primer to template 3. Perform synthesis reaction with a DNA polymerase enzyme in the presence of dNTPs and small amount of "chain terminator"
pUC8 plasmids What do pUC8 plasmids contain?
• Ampicillin resistance gene • lacZ' gene, codes for part of the β-galactosidase enzyme (splits lactose into galactose and glucose) • Multiple cloning site (polylinker) within the lacZ' open reading frame
minisatellites?
• Repeat unit is a few tens of nucleotides in length • Variation generated by unequal crossing over during meiosis • Are non-randomly distributed around the genome (tend to be near chromosome ends, e.g., telomeric repeats)
blue-white screening
• When using pUC18 plasmid, transformed cells are plated on agar containing antibiotic Ampicillin and X-gal • cells containing plasmid will survive in the antibiotic • cells containing pUC8 without DNA fragment insert will express lacZ' gene and produce β-galactosidase, the enzyme that can splits X-gal into its component sugars, one of which is blue • Therefore, cells containing pUC8 without insert are blue, those with inserted DNA are white
