MOLBIOL 4RR3 TEST 2

DNA amplification

PCR

• selective DNA amplification

• prior seq infor = required to design 2 primers specific for the target

• primers + target seq + heat stable DNAP + dNTP = amplification of a dna target fragment

why is heat stable polymerase required for PCR?

rxn made of 3 cycles

• denaturation - 93-95°C

• reannealing - 50-65°C

• dna synthesis - 70-75°C

specificity of amplification and primer design

• nested primers

  • initial amplification useed as a target source for a second rxn

• hot-start pcr

  • leave the enzyme or primers until the first denaturation step

  • lowers background resulting from nonspecific binding of primers

• touchdown pcr

  • annealing temp = lowered gradually

  • to increase specificity early in amplification

PCR pros and cons

Applications of PCR

• for screening uncharacterized mutations

• permits rapid genotyping for polymorphic markers

• assaying for known mutations

• allelic discrimination by size or susceptibility to RE

• allelic discrimination by susceptivity to an artificially introduced RE site

• allele-specific PCR = primers are designed so as to discriminate bw target DNA seqs that differ by a single nt in the region of interest

• degenerate oligonucleotide-primed PCR

• linker-primed PCR - ligation adapter pcr

• anchored pcr - target-specific primer + universal primer

Cell-based DNA cloning - VECTOR SYSTEMS

Plasmids

Cosmid vectors

Vectors based on bacteriophage P1 and F factor plasmids

• bacterial artificial chromosomes (BAC) vectors

• bacteriophage P1 vectors and PACs

• yeast artificial chromosomes (YACs)

Plasmids

Origin: natural bacterial plasmids

Important features;

• multiple cloning site (MCS)

• antibiotic resistance gene

• screening system for recombinant plasmids

DNA inserts: small fragments

Cosmid vectors

Structure: plasmid containing bateriophage λ cos sequence

Important features:

• combine plasmid and phage features

• used to clone larger DNA fragments

DNA inserts: 30-44 kb

Bacterial Artificiakl Chromosomes (BAC) vectors

origin: E. coli F factor - fertility plasmid

important features:

• low-copy-number replicon

• provides greater stability for large inserts

• can maintain large DNA fragments

DNA inserts: up to 300 kb

bacteriophage P1 vectors

origin: bacteriophage P1

important features:

• P1 components incorporated into a circular plasmid

• P1 packages its genome inside a protein coat

DNA inserts: large fragments

P1-derived artificial chromosomes PACs

structure: combination of P1 and F-factor systems

important features:

• Combines features of both systems

• Used for cloning large DNA fragments

DNA inserts: large fragments

yeast artificial chromosomes (YACs)

origin: artificial yeast chromosome

important features:

• contains essential chr components

1. centromeres → required for disjunction of sister chromatids in mitosis

2. telomeres → required for complete replication and protection of the chromosome ends

3. autonomous replicating seq elements → required for autonomous replication of chromosomal DNA, thought to act as specific rep origin

• allow replication and stable maintenance of large DNA fragments

DNA inserts: large fragments

“4 SHORT SEQS REQUIRED → 2 TELOMERERS, 1 CENTROMERE, 1 ARS”

Nuclear acid hybridization

• ssDNA binds to its complementary dna seq → forms dsDNA

• purpose: uses a labeled nucleic acid probe to find matching DNA and RNA molecules in a mixture of unlabeled nucleic acid mols

Preparation of nucleic acid probes

• conventional dna probes

  • labeled by adding labeled dNTPs

  • isolated by cell-based dna cloning or by PCR

• rna probes

  • generated from DNA within specialized plasmid containing a phage promoter sequence

• oligonucleotide probes

  • short ssDNAs

NA hybrization - Factors affecting dna strand separation

1. strand length

   • longer strands = more H+ bonds

   • require more energy to separate them

2. base composition - GC content

   • GC pairs = 3 H bonds

   • AT pairs = 2 H bonds

   • higher GC content = stronger bonding = harder to separate

3. chemical envr

   • Na+ ions - stabilize DNA strands

   • formamide and urea - destabilize dna strands = easier to separate

DNA and RNA labeling

1. during in vitro synthesis

   1. labeling of DNA

      • nick-translation

      • random primed labeling/PCR mediated labeling

   2. labeling of RNA

      • in vitro transcription system

2. end-labeling

   • involves the addition of labeled group to the terminal nts

southern blot hybridization (SBH)

• target DNA = digested with one or more restriction endonucleases

• size-fractionated by agarose gel electrophoresis

• denatured and transferred to NC for hybridization

• immobilized ssDNA seqs = allowed to associate with labeled ss probe DNA

Southern Blotting Hybridization - Application

1. Restriction enzyme (RE) mapping

   • detects pathogenic point mutations

   • identifies changes in RE recognition sites

2. Restriction fragment length polymorphism (RFLP)

   • detects differences in dna fragment lengths

   • mutations can create or remove RE sites, changing fragment sizes

3. VNTR-basex RLFPs (DNA fingerprinting)

   • detects differences in the number of repeated DNA seqs

   • VNTR = variable number tandem repeats

   • also called hypervariable minisatellite DNA (5-64 bp)

   • DNA probes detect these differences

4. Gene deletion detection

   • restriction mapping can identify missing DNA sections

Tissue in situ hybridization

a labeled probe is hybridized against RNA in tissue sections

Colony blot and plaque lift hybridization

1. grow colonies → bacteria containing plasmids are grown on an agar palte

2. transfer → colonies are transferred onto nitrocellulose filter by direct contact

3. dna denaturation → colonies treated with alkali to separate dsDNA into ss

4. hybridization → a labeled nucleic acid probe binds to complementary DNA seqs

5. detection → filter is washed, dried, and exposed to x-ray film (autoradiography) to identify positive colonies

Gridded high density arrays of DNA clones

• uses robotic gridding devices

  • automatically transfer DNA clones from microtiter plates onto a membrane

• each dna clone = placed at a specific, predetermined linear position (coordinate)

• purpose: allows rapid and efficient screening of large DNA libraries to identify specific dna clones

DNA microarray technology

Purpose: allows large-scale, rapid and automated DNA hybridization

Main advantage: miniaturization and automation allow thousands of DNA samples to be analyzed at once

Three main types:

1. DNA clone microarrays → dna clones are placed onto a surface using microspotting

2. DNA probe microarrays → dna probes are placed onto a surface using microspotting

3. Oligonucleotide microarrays → short DNA seqs are synthesized directly on the surface (in situ)

Application: identify rare target seqs, analyze gene exp

Detection: laser scanning detects fluorescent signals from hybridized probes

CRISPR-Cas9 Technology

CRISPR → clustered regularly interspaced short palindromic repeats

Discovery

• identified by francis mojica, 1996

Function

• acts as a primitive immune system in bacteria by storing DNA seqs from previous viral invaders

Cas9 protein

• recognizes matching DNA seqs and cuts the target DNA

Potential applications for CRISPR-Cas9 technology

1. human therapeutics → treat genetic diseases by correcting or modifying faulty genes

   • sickle cell disease

   • beta-thalassemia

   • duchenne muscular dystrophy

   • cancer

2. nonhuman apps

   • agriculture → improve crops and livestock

   • livestock → modify animals to improve desirable traits

   • industrial biotechnology → produce useful biological products

3. gene editing processes

   1. guide rna directs cas9 toa specific dna seq

   2. cas9 cuts target dna

   3. cell repairs dna → allow the gene to be disrupted or corrected

First CRISPR-edited babies

Researcher: he jiankui

November 2018: announced the birth of the world’s first genetically edited babies → lullu and nana

target gene: CCR5, codes for a protein that HIV uses to enter certain immun cells

goal: disable the CCR5 gene to make the babies more resistant to HIV infection

outcome: gene was deited in human embryos = successful pregnancy and birth of twins

RAISED MAJOR ETHICAL CONCERNS

disabling CCR5 does not guarantee complete immunity to HIV

long term effect unknown

Single cell RNA sequencing (scRNA-seq)

Purpose: measures gene exp in individual cells to identify differences bw cell types

Workflow

1. collect tissue

2. dissociation → break down tissue into individual cells

3. rna extraction

4. reverse trancription → convert RNA into cDNA

5. amplification

   • PCR to amplify cDNA

   • In vitro transcription → to produced amplified RNA

6. sequencing library preparation

7. sequencing → determine rna-derived seqs

8. gene expression profiling → analyze which genes are expressed in each cell

9. clustering → group cells with similar gene expression patterns to identify different cell types

Constructing trancript maps

Purpose: identify genes and expressed regions within cloned gDNA

Methods:

1. Hybridization to RNA/cDNA

   • DNA clone is hybridized against a northern blot containing a panel of mRNA isolated from different tissues

   • positive signal = cloned DNA contains a gene expressed in that tissue

2. Zoo blot hybridization

   • compares DNA seqs across different species

   • coding regions are highly conserved between species

   • noncoding regions are less conserved

3. CpG island identification

   • short (~1 kb), GC-rich, hypomethylated DNA regions

   • found near the 5’ end of genes

   • approximately 56% of genes are associated with CpG islands

4. Exon trapping

   • exons can be identified in cloned gDNA

   • dna is inserted into an expression vector (eg pSPL3)

   • vector is introduced into eukaryotic cells

   • RT-PCR used to detect and retrieve trapped exons

5. transcript maps

   • RNA-seq data or ESTs (expressed seq tags) are mapped to specific genomic regions

   • mapping can be done using computer analysis or PCR

6. computer analysis

   • homology searches identify similar DNA seqs

   • exon prediction programs identify potential exons

Expression Studies at Different Levels

Data integration

General Nucleotide and Protein Sequence Repositories

Eukaryotic genome database

Program for sequence homology search

Organization of the human genome

Nuclear genome

• 24 linear ds DNA molecules, ~3200 mB, ~30000 genes

• ~45% transposon-based repeats

• ~44% other non-conserved

• ~6.6% heterochromatin

• ~3% highly conserved (other)

• 1.5% highly conserved (coding)

Mitochrondrial genome

• 1 circular double-stranded DNA molecule, 16.6 kb, 37 genes

• ~93% highly conserved (coding)

• ~5% highly conserved (other)

• <2% other non-conserved

Human Nuclear DNA categorization

1. single sequence DNA/low copy DNA

   • ~45% of total genome

   • cds = ~1.5% of total genome

   • DNA from introns = 53.5%

2. intermediate repeated DNA

   • present at between 10² and 10^5 copies per genome (~45% in total)

3. Highly repetitive DNA

   • present at up to 10^6 copies per genome (~10% of the total)

Human genes

• about 30-35000 in total

• some genes (ie histone genes) do not contain introns

• alpha globin gene

  • size: 0.8 kb

  • introns count: 3, account for 30% of total genomic locus

• dystrophin

  • gene defective in DMD

  • size: 2.4 Mb

  • introns count: 79, account for 99.4% of total genomic locus

  • gene mean size: 27 kb

Gene length and exon content

Overlapping genes within the class III region of HLA complex

Genes within genes: Neurofibromatosis type 1 gene (NF1)

intron 26 of NF1 gene contains 3 interal two-exon genes

Gene families

• arise by gene duplication and divergence from an ancestral gene

• because a family has multiple copies, a loss of function in one may be tolerated

• led to evolution of pseudogenes - non-functional copies

Clustered and interspersed multigene families

Pseudogenes

defective gene copies found in gene families

1. unprocessed pseudogenes in a gene cluster

   • tandem gene duplication, inappropriate termination codons

2. truncated genes and internal gene fragments

   • eg. class I HLA

3. nonprocessed pseudogenes in a dispersed gene family

   • eg. NF1 gene fragments over 7 chrs

4. processed pseudogenes in a dispersed polypeptide-encoding gene family

   • cDNA + oligo dA/dT. copied by retroptransposition

5. processed pseudogenes in an RNA-encoding gene family

   • retrotransposition from RNAP III transcripts

Processed pseudogenes

1. gene transcribed into mRNA

2. introns are spliced out = mRNA has poly-A tail in 3’ end

3. reverse transcriptase = RNA to cDNA

4. cDNA gets pasted into the genome at random spot

5. cell repairs and fills in the dna, leaving short repeated sequences on each side

Non-protein coding genes

bulk of cellular RNA consists of rNA and tRNA required for mRNA translation

additional functions of noncoding RNAs

1. snRNA invovled in spliceosome function

2. snoRNA - control site specific base modifs in rRNA and U6 snRNA

3. 7SL rna molecule - forms part of the signal recognition particle required for the translocation of proteins across the ER

4. miRNAs - 22 nts long can function as antisense regulators of genes to inhibit translation

5. lncRNA - rna molecules >200 nts, can modulate gene expression

Genes

dna sequence that contributes to the phenotype of an organism in a way that depends on its sequence

RNA polymerases

Prokaryotes (E. coli)

1. one RNAP transcribes all genes

2. control region has two promoter elemts

   • at -35 and -10, upstream of the transcription start site (+1)

Eukaryotes

3 RNAPs

1. Pol I - transcribes rRNA

2. Pol II - transcribes polypeptide-encoding genes (mRNA) and some RNAs

3. Pol III - transcribes tRNAs, 5S rRNAs, and small RNAs

Promoter

• typical eukaryotic gene has a promoter region, extends about 200 bp upstream of the TSS (nt + 1)

• promoter contains one or more of these elements:

  • enhancers

  • response elements

  • silencers

• most human promoters have TATA box

  • centered around position -30 relative to the TSS

  • binds general transcription factors, designated TFIIX (X = placeholder for specific factor)

Pol II transcription initiation

1. TFIID (containing TBP) binds the TATA box upstream of the TSS

2. TFIIB joins the complex

3. RNAP II arrives together with TFIIF and other TFs

4. TFIIE and TFIIH are then added = complete initiation complex

5. the nts (ATP, UTP, CTP, GTP) are added

6. RNA synthesis begins

7. phosphate groups added to the tail of RNAP II

8. RNAP II moves along the DNA as RNA is made, leaving some general factors behind at the promoter

Transcription factors for transcription by RNAP II

Regulatory elements within the promoter

• GC box (GGGCGG → binds the TF SP1

• CAAT box (GGCCAATCT) → binds the TFs CTF and NF1

• Oct box (ATTGCAT) → binds the TFs Oct-1 and Oct-2

• Enhancers → operationally distinguished from promoters by 3 criteria

  • located a considerable distance from the TSS

  • located upstream or downstream

  • action not dependent on orientation

Eukaryotic structural gene

DNA looping

a type of gene regulation

brings proteins bound to enhancers or silencers into direct contact with proteins bound to the promoter

Response Elements

What? induce genes in response to signals

Where? within promoter region, closely upstream of the promoter or in more distant locations

Examples

• cAMP response element (CRE)

• serum response element (SRE)

• heat-shock response element (HRE)

Silencers

elements that bind certain proteins, resulting in negative control of gene expression

Housekeeping genes

• expressed in most tissues most of the time

• responsible for functions likely to be necessary in any cell

cAMP response

1. a hormone binds its receptor on cell mem

2. receptor activates a G protein (⍺ subunit separates from the β and γ subunits)

3. G protein activates adenylate cyclase → converts ATP into cAMP (+PPi)

4. cAMP binds the regulatory subunits of PKA → releases the active catalytic subunits (before PKA is inactive complex of 2R and 2C subunits)

5. free C subunits move into the nucleus

6. C subunit phosphorylates CREB

7. phosphorylated CREB activates target genes that contain a CRE

Transcrption

1. RNAP transcribes the DNA template

   • mRNA already has a 5’ cap and polyadenylation (AAUAAA) signal appears in the transcript

2. a spscific endonuclease cleaves the mRNA downstream of the AAUAAA signal

3. poly A polymerase adds adenine residues to the new 3’ end, using ATP (releasing PPi)

4. result: mRNA with a 5’ cap, the AAUAAA signal, and a poly-A tail (~100-250 adenine res) at 3’ end

   

Cap formation

as soon as transcription starts

• 5’ end of nascent mRNA molecule is covalently modified to form a cap

• by addition of 7-methylguanosine to 5’ nt of nascent mRNA in a 5’-5’ triphosphate linkage

• cap is essential for initiation of translation

Transcription termination

1. sequence AAUAAA in the nascent RNA triggers termination a few hundred bps downstream

2. an endonuclease cuts the RNA downstream of this signal

3. 5’ → 3’ exonuclease degrades the RNA still attached to the polymerase

4. transcription stops when exonuclease meets the polymerase

Polyadenylation

RNA is cleaved about 20 bp downstream of the AAUAAA signal

3’ end is then covalently modified by adding 100-200 adenine residues = poly-A tail

One gene, more than one protein

alternative transcription and processing of genes can generate multiple protein products

1. alternate promoters → generate tissue-specific isoforms

2. alternative splicing and polyadenylation → human genes are prone to both

3. RNA editing → specific cytosine or adenine residues are substituted by deamination (C→U editing in APOB gene)s

Dystrophin gene - multiple promoters

has at least 7 promoters that drive tissue- and cell type-specific expression

each promoter has its own first exon, which is spliced onto the shared downstream exons (numbered 2 to 79)

Types of alternative splicing

A. exon skipping (cassette exon)

B. alternative 5’ splice site - exon is included in a longer or shorter form, depending on which 5’ splice site is used

C. alternative 3’ splice site - same idea, but with 3’

D. intron retention 0 blue region bw the two pink exons = either spliced out as an intron or kept in the matuer mRNA

E. mutually exclusive exons - blue exon or brown exon included, never both

Neuroxin-3 transcript in CNS - alternative splicing

combines alternate promoters with alternative splicing, so one gene can produce a very large number of different transcripts and protein isoforms in the CNS

Tissue-specific RNA editing of the APOB gene

Gene

• APOB has a large exon 26 → contains a CAA codon at nt 6666

Liver

• no editing occurs, CAA codon is read normally

• translation continues to downstream stop codon = full length ApoB100 protein

Intestine

• cytidine deaminase APOBEC1 edits the mRNA

  • change C → U at p6666

  • turns CAA into UAA = premature stop codon

  • translation stops early = shorter ApoB48

Cholesterol cycle

intestine → liver (dietary route)

• dietary lipids absorbed in the intestine → packaged into chylomicrons, travel to the lievr

• in the enterocyte, MTP helps load lipid onto ApoB48 to form chylomicron

liver → periphery (VLDL → LDL)

• in hepatocyte, MTP loads lipid droplets onto ApoB100 to form VLDL

• VLDL → LDL, delivers cholesterol to peripheral tissues

periphery → liver (reverse cholesterol transport, HDL)

• pre-beta HDL picks up cholesterol from peipheral cells

• matures into HDL3, then HDL2

• HDL2 returns to the liver, where cholesterol can be removed in bile

• CETP transfers lipids bw HDL and VLDL/LDL

CpG Islands

DNA can be modified by methylation of cytosine to form 5-methylcytosine

occurs at the dinucleotide CpG

CpG

• occurs less frequently in the genome, because

  • accidental deamination of cytosine produces uracil

  • whereas deamination of 5'-methylcytosine produces thymine

• uracil not found in DNA, replaced with cytosine by glycolase

• thymine is mispaired with G res

DNA mehtylation

generally associated with repression of transcription

CpG islands are about 1kb in length

tend to extend over promoters of expressed genes

About 56% of human genes are estimated to be associated with such seqs

Changes in DNA methylation during mammalian development

Germ line (before fertilization)

1. primordial germ cells (PGCs) start with a high degree of methylation

2. around gonadal differentiation → erasure of parental epigenetic settings (methylation drops at low level)

3. as germ cells develop → de novo methylation and establishment of imprinting marks occur

   1. producing methylated sperm and egg

After fertilization

1. early embryo → demethylation

2. blastocyst stage → methylation level is lowest

After blastocyst

• de novo methylation begins again in somatic cells, then maintenance methylation keeps the pattern in adults.

Placenta and yolk sac (trophoblast lineages) are methylated too, but less than the embryo proper.

Some cells become PGCs (primordial germ cells), and the cycle starts over.

Chromatin conformation - ChIP

1. Crosslink proteins to DNA, lyse the cells, and isolate the chromatin.

2. Fragment the chromatin and mix it with an antibody specific for just one protein.

3. Precipitate the antibody-bound protein-DNA complexes.

4. Reverse the crosslinking, degrade the protein, and purify the DNA fragments.

5. Analyze the purified DNA in one of these ways:

• Standard ChIP: test specific target regions.

• Genomewide ChIP:

  • ChIP-chip: hybridize the fragments to a microarray.

  • ChIP-PET or STAGE: tag the sequence fragments.

  • ChIP-seq: sequence the fragments.