Lecture 24: DNA technology

Nucleotides absorb _____ _____

260nm light

Chargaff determines that the ratio of A:T and G:C (purines: pyrimidines) is always

1

Nucleotide light absorption can determine

DNA purity

Characterizing Properties of DNA sequences- two DNA strands separated by _____ them apart with heat

melting

Melting temperature (tm)

specific temperature at which half of the DNA strands separate

Denaturation graph at 0

none are denatured

Denaturation graph at 100

all are denatured

As temperature increases, dna begins to

denature

Melting temperature (tm) increases as the number of __-___ pairs increases in DNA sequence

G-C

It takes more energy to separate __-____ DNA pair

C-G

Recombinant DNA

DNA formed artificially by combining constituents from different organisms

Study the genome in fragments

digest genome with ____ _____into random pisces and ligate them into ____ ____

Steps for recombinant DNA

1) cloning vector is cleaved with resirtcion nucleases 2) DNA fragment of interested is obtained by cleaving chromosome with the same restriction endonuclease 3) fragemenst are ligated to the prepared cloning vector

DNA is cleaved at specific sequences by

restriction endonucleases

Does restriction endonuclease cleave randomly

NO

What do restriction endonuclease cut

phosphodiester linkage

Restriction endonucleases often bind and cut the phosphodiester backbone at ______ _____

palindromic sequences

Two types of DNA cleavage

sticky ends or blunt ends

Sticky ends

some enzymes leave overhands

Example of an enzyme that leaves an overhang

hindlll digest

Blunt ends

enzymes cut at the same site on both strands

Plasmids

circular pieces of DNA that only contain a few genes

Plasmids are used primarily by

prokaryotes

Plasmids contain more or less base pairs that the genome

LESSS

Selecting for recombinant plasmids- plasmids include a ___ ___ that commonly confer ____ ___

selection marker that commonly confers antibiotic resistance

Plasmids are transformed into

bacteria

Plasmids teaching bacteria how to get rid of

an antibiotic)

Term for lawn, bacteria growing all over the plate, no idea what bacteria picked up the plasmid, does not contain any antibiotic

Nonselective

Ampicillin

every cell in colony contains plasmid

What express resistance to ampicillin

only cells that take up the plasmid

Polymerase chain reaction (PCR)

DNA replication in a test tube, clone specific pieces of DNA

Polymerase chain reaction steps

1) melt

After 20 cycles, target sequence is amplified_____ fold

10^6

Design primers engineered with a ____ enzyme cute site so your PCR fragment can be cut and ligated into a plasmid

restriction

Agarose gel electrophoresis direction of DNA movement

larger framenets (cathode -) -> smaller fragments (anode +)

Remember- DNA is normally _____ charged

negatively charged (due to backbone)

Photo

Ethidium bromide

Ethidium bromide

common stain for DNA

Ethidium Bromide function

intercalates between bases, causing it to fluoresce under UV excitation

Complementary DNA (cDNA)

generated from mature mRNA

Reverse transcriptase

an RNA dependent DNA polymerase

cDNA synthesis steps

lyse cells and purify mRNA, hybridize with oligo dT primers, reverse transcription, degrade RNA with Nase H, synthesis of complementary DNA with DNA polymerase

Purification of recombinant proteins- some plasmids utilize a ____ ____ to control overexpression of proteins

inducible promoter

Epitope tag

add antibody recognition to a protein for purification and visualization, tagged to be identified by antibodies

Recombinant DNA can create

fusion proteins

Example of tag fused to proteins

glutathione-S-transferase (GST)

Fusion protein is eluded in the ____ columns

later

Fusion proteins can be used for

affinity chromatography or in aiding in visualizing cellular location

Example of fusion protein used for aid in visualizing cellular location

green fluorescent protein (GFP)

Green fluorescent protein (GFP)

emits light upon excitation

GFP variants are available in what colors

many different colors

Recombinant proteins can function as

MEDICINE

1921-banting and best discover ____ as a cure for diabetes

insulin

Insulin was originally produced from

cows and pigs

Manipulating the sequence of recombinant DNA through what process

oligonucleotide-directed mutagenesis

oligonucleotide-directed mutagenesis- Manipulating the sequence of recombinant DNA only works for

plasma DNA

oligonucleotide-directed mutagenesis example

asp in the active site -> switch to ala

oligonucleotide-directed mutagenesis steps

1) denature plasmid and anneal oligonucleotide primers with mutation 2) use DNA polymerase to extend and incorporate the mutagenic primers 3) digest nonmutated parental DNA template with methylation-specific nuclease, and anneal newly synthesized strands 4) transform dsDNA into cells. Cell repairs nicks in mutated plasmid

CRISPR/Cas9

enables precise targeting of DNA sequences

Natural CRISPR-Cas mechanism steps

1) virus invades bacterial cell 2) new spacer is derived from virus and integrated into CRISPR sequence 3) CRISPR DNA is formed 4) CRISPR RNA guides molecular machinery to target and destroy virtual genome

Guide RNA

targets specific DNA sequences

CAS

CRISPR Associated nuclease

Nuclease

domains cleave target DNA

Small palindromic repeats were acting as

guide RNA

Steps for CRISPR

1) isolate patients hematopoietic stem and progenitor cells (HSPC) 2) edit cells in culture with CRISPR 3) re-establish stem cell population in bone marrow

CRISPR has been able to help cure

sickle cell disease (previous that incurable due to being genetic)

DNA sequencing uses

Sanger sequencing

ddNTP

DiDeoxy nucleotides, terminate replication because they lack a 3’ -OH

Sanger sequencing originally used

radioactive nucleotides

Then sanger sequencing used

fluorescent markers, read by a laser beam

Sanger sequencing-Reaction 1

1 sequence read, know something about your sequence (primer)

Next generation of sequencing

sequencing by synthesis

In the next generation of sequencing what happens

add adaptors → attach to flow cell -> sequencing -> signal scanning

a picture of the entire flow cell is taken after…

each fluorescent base is added

Sanger sequencing requires

A PRIMER

Next generation of sequencing

sequencing by synthesis

Sequencing by synthesis

Synthesis the complementary strand and reading as you are synthesizing

Each spot is a ___ _____ and the color changes each image as ___ ____ are added to the _____ ____

DNA fragment and the color changes each image as new bases are added to the complementing strand

Overlapping reads are built… and mapped onto….

into a contiguous sequence (contigs) and mapped onto a reference genome

Measuring changes in gene expression- quantitative real time-

Polymerase chain reaction

utilize fluorescent probes to monitor amplification

In qRT-PCR you isolate what and convert what

Isolate mRNA and convert DNA

Chain reaction, meaning it grows

exponentially

Amount of DNA produced should correlate with

mRNA levels

CT

cycle threshold, marks early exponential growth

Signaling graph- shift to the Left- more/less template DNA

more

Signaling graph- shift to the Right-more/less template DNA

less

Lower CT value indicates higher

mRNA levels (higher expression)

Microarray analysis

allows you to compare expression across an entire transcriptome

NOTE- microcray is a ________ of two different conditions of a cell

COMPARISON

Current approaches utilize ____ to get highly sensitive transcriptome analysis – even the transcriptome of a single cell

NGS

Human genome ⅓ of proteins

we still do not know what they do

Polymorphism

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

Polymorphism in pigment production genes lead to ___ ____ in jaguars

different phenotypes

We are all human, yet ____ _____ variation leads to distinct appearance

slight genetic variation

Genome analysis identities

single nucleotide polymorphism (SNPs)

Haplotypes are groups of

multiple SNPs

Tag SNPs are

unique and define the halotype

sequencing technology helps identify _______ and more accurately draw _____ trees

haplotypes; phylogenetic trees

More lactose in tolerant in gernatine regions