DNA Technology

5 key concepts

1. Synthesis of recombinant DNA

2. PCR to amplify specific sequences of DNA

3. Recombinant DNA can be used to form protein fusions

4. DNA sequences can be specifically manipulated

5. DNA sequencing technology has increased in speed and fidelity

Recombinant DNA

• Restriction enzymes

• Plasmid Vectors

PCR based technologies

• Oligonucleotide primers

Recombinant fusion proteins

• Epitopes

• Affinity tags

• GFP

Manipulating DNA sequence

• Quick Change

• CRISPR/CAS9

Measuring gene expression

• Microarray

• RT-PCR

DNA sequencing technology

• Sanger

• Sequencing by synthesis

identifying and purifying dna

Chargoff determines that the ratio of A:T and G:C

• (Purines:Pyrimidines) is always 1

Characterizing Properties of DNA Sequences

Remember: It takes more energy to separate C – G pair

1. Melting Temperature (tm) is____________

   1. increases as __________________ in DNA sequences

1. the specific temperature at which HALF of the DNA strands separate

   1. INCREASES as the number of G–C pairs increases in DNA sequences.

Recombinant DNA

Study the genome in fragments: how?

DNA formed artificially by combining constituents from different organisms.

Digest genome with restriction enzymes into random pieces and ligate them into prokaryotic plasmids.

DNA is cleaved at specific sequences by

restriction endonucleases

Restriction endonucleases (restriction enzymes) often bind and cut the phosphodiester backbone at ___________________

palindromic sequences.

Some enzymes leave overhangs (____________) example?

sticky ends, HindIII digest

Other enzymes cut at the same site on both strands (________) example?

blunt ends e.g. EcoRV digest

Plasmids are

circular pieces of DNA that only contain a few genes.

Selecting for recombinant plasmids

Plasmids include a selection marker that commonly confers ___________________________

Plasmids are transformed into __________.

Only cells that take up the plasmid_____________________.

antibiotic resistance

bacteria

express resistance to ampicillin

PCR is? steps? MAE

1. Melt: Heat up to separate strands

2. Anneal: Short oligonucleotide primers bind

3. Elongation: DNA polymerase synthesizes new DNA strand.

Design primers engineered with a restriction enzyme cut site so THAT

your PCR fragment can be cut and ligated into a plasmid.

Agarose gel electrophoresis

________ _______ is a common stain for DNA

what does it do?

DNA is normally __________ charged

Ethidium bromide

• It intercalates between bases, causing it to fluoresce under UV excitation.

  • Remember: DNA is normally negatively charged

1. BamH1 cleaves the following sequence. Does this result in a blunt end or sticky end?

2. Bcl1 cleaves the following sequence. Write the sequence of the overhang after digestion.

3. How many fragments will result from the cleavage of the following sequence with BamHI and Bcl1?

Cloning the coding sequence of genes

DNA contains all introns and exons:

• How can you create recombinant DNA with just exons?

Complementary DNA (cDNA) is generated from mature mRNA.

steps for cDNA synthesis

• Remember: Reverse transcriptase is an RNA-dependent DNA polymerase.

Purification of recombinant proteins

Some plasmids utilize an __________________ to control overexpression of proteins.

Epitope tags add ________________ to a protein for ___________ and _____________

Some plasmids utilize an inducible promoter to control overexpression of proteins.

Epitope tags add antibody recognition to a protein for purification and visualization.

Recombinant DNA can create ________________ for what?

fusion proteins for affinity chromatography

Recombinant DNA can create _______________ that aid in what?

fusion proteins that aid in visualizing cellular location

Green Fluorescent Protein (GFP) ____________________-

emits light upon excitation.

Manipulating the sequence of recombinant DNA

what is oligonucleotide directed mutagenesis? end goal?

Asp in the active site → Switch to Ala

Codon:

• GAU → GCU

• GAT → GCT

CRISPR/Cas9 enables what

name three purposes of Cas9 complex

precise targeting of DNA sequences

Clustered Regularly Interspaced, Short, Palindromic Repeats

1. scans genome for target sequence

   1. nonhomologous end joining = gene mutation

   2. recombination with fragment = homology-directed repair

2. gene knockout through frameshift → insert stop codon

   1. double strand break repair mechanisms

3. transcriptional repression/activation

CRISPR ASsociated nuclease (CAS) do two things

1. Guide RNA targets specific DNA sequences

2. Nuclease domains cleave target DNA.

Gene editing with CRISPR (3 steps)

1. Isolate patient’s Hematopoietic Stem and Progenitor Cells (HSPC) 

2. Edit cells in culture with CRISPR

3. Re-establish stem cell population in bone marrow

Sanger Sequencing

what is ddNTP? why does it terminate replication?

DiDeoxy Nucleotides (ddNTP) terminate replication because they lack a 3’ –OH (3’ H CAN’T act as a nucleophile for phosphodiester bond formation)

• 1 reaction → 1 sequence read

  • know what about your sequence?

• Know something about your sequence (primer).

Next Generation Sequencing – Sequencing by Synthesis

A picture of the entire flow cell is taken after each fluorescent base is added.

Each spot is a _____________ and the color changes each image as new bases are added to the __________________

Overlapping reads are built into a ____________________________ and mapped onto a reference genome.

DNA fragment

complementing strand.

Overlapping reads are built into a contiguous sequence (contigs)

Measuring changes in gene expression

Quantitative Real-Time – Polymerase Chain Reaction (qRT-PCR) does what?

utilizes fluorescent probes to monitor amplification.

steps of qRT-PCR (5)

• Isolate mRNA and convert to DNA

• Amount of DNA produced should correlate with mRNA levels.

Lower Cycle Threshold (CT) value

indicates higher mRNA levels (higher expression)

Measuring changes in gene expression

Microarray analysis allows you to ________________________________

compare expression across an entire transcriptome.

• Note: Comparing two conditions of a cell. (well fed vs starved, control vs stressor, etc.)

Current approaches utilize NGS to get ____________________ analysis

highly sensitive transcriptome analysis – even the transcriptome of a single cell!

Polymorphisms:

Small variations in gene sequence that lead to changes in phenotype and variation in populations.

Genome analysis identifies ____________________?

Haplotypes are ______________________

Single Nucleotide Polymorphisms (SNPs)

groupings of multiple SNPs

Tag SNPs are _______ and __________

Sequencing technology helps _____________ and more accurately _______________

unique and define the haplotype.

identify haplotypes and more accurately draw phylogenetic trees.

• DNA sequencing will allow the ________________________ and ____________________

• identification of unique populations within cancerous cells and prescribe more effective treatments.