biotechnology recombinant DNA and genomic analysis (1)

Cut long strings of DNA into fragments with restriction enzymes and separate them by gel electrophoresis 2.. Isolate, amplify, and purify fragments through molecular cloning 3.. 9.4 Gel

Recombinant DNA and

allied methods

1. Cut long strings of DNA into fragments with restriction enzymes and separate them by gel electrophoresis

2. Isolate, amplify, and purify fragments through molecular cloning

3. Use purified DNA probes to identify similar sequences in libraries of clones or mixtures of DNA or RNA molecules

4. Rapidly isolate and amplify previously defined genomic or mRNA sequences through the polymerase chain reaction (PCR)

5. Determine the precise sequence of bases within isolated DNA fragments

1 Fragmenting complex genome into pieces for analysis

Restriction enzymes

fragment the genome

at specific sites.

Fig 9.1

Restriction enzymes

 Restriction enzymes

cut DNA molecules at specific locations

produce either blunt or sticky ends (cohesive ends)

Fig 9.2

Different REs produce fragments of different lengths

 The number of base

pairs a restriction

enzyme recognizes

determines the average

distance between sites

and the size of

fragments produced.

 Probability that a

four-base recognition site

will be found in the

genome =

¼ x ¼ x ¼ x ¼ = 1/256.

Fig 9.3

Time of exposure to a RE helps determine fragment size

The genetics use RE to produce DNA fragments of a particular length:

 Complete digest: cutting at every recognition site

 Partial digest: cutting by controlling amount of enzyme

and time the DNA is exposed to the RE when researches need the DNA fragments with expected lengths

Partial digests are used so enzymes cut only a subset of the total number of

recognition sites in a genome

Fig 9.4

Gel electrophoresis separates DNA fragments according to size

Preparing an agarose gel for electrophoresis

Fig 9.5a

Loading DNA fragments onto an agarose gel and performing electrophoresis

Gel electrophoresis separates DNA fragments

according to size.

Fig 9.5a

Visualizing the DNA fragments on the agarose gel

Gel electrophoresis separates DNA fragments according to size

Fig 9.5a

Polyacrylamide gels separate very small fragments of DNA and agarose

gels separate larger fragments

Visualizing the DNA fragments on the agarose gel

Fig 9.5b

Restriction maps can be inferred from DNA fragments cut with two enzymes providing a roadmap to DNA fragments and virus genomes

Steps to determine the order of restriction sites along

a DNA fragment:

 Divide a purified preparation of cloned DNA into three aliquots

 Cut one aliquot with EcoRI, one with BamHI, and the

third with both enzymes

 Separate fragments by gel electrophoresis

 Determine the size by comparing to a size standard

 Use a process of elimination to derive the only arrangement that can account for the sizes of the fragments obtained in all three aliquots

Restriction mapping

Fig 9.6

2 Cloning fragments of DNA

Genomes of animals, plants, and microorganisms are too large to analyze using gel electrophoresis and restriction mapping.

Cloning is a means

 to identify a specific DNA fragment within the genome,

 purify away all of the other fragments,

 and amplify it, making many identical copies of the fragment

Such a fragment can then be analyzed by restriction mapping and DNA sequencing.

Two strategies to purify and amplify individual

fragments

Polymerase chain reaction

Purification and amplification of previously

sequenced genomic regions

Molecular cloning

Purification and amplification of previously

uncharacterized DNA

Cut DNA and insert fragments of specific sizes into

vectors

Transport vector-insert molecules into living cells that

make many copies

DNA clones are any amplified set of purified DNA

molecule

2.1 Step 1 of molecular cloning

 Ligation of fragments into cloned vectors creates recombinant DNA molecules

Sticky ends facilitate recombinant DNA fabrication.

 Cutting the vector and DNA fragments generates

complimentary sticky ends that increase the efficiency of

ligation between the vector and insert DNA

Step 1 of molecular cloning

2.2 Step 2 of molecular cloning

 Host cells take up and amplify vector-insert

recombinants.

 Transformation – vectors carry insert DNA into cells

 Recombinant DNA molecules are added to a suspension

of competent E coli

 Cells are heat-shocked or shocked with a high-voltage

electric shock (electroporation)

 Transformants are identified

 Ampicillin resistance – cellular clones are colonies that

represent each viable plasmid containing bacterial cell

 Plasmid-insert transformants are identified

 b-galactosidase selection

 Cells that turn blue DO NOT have an insert b-gal

gene is intact

 Cells that are white DO have an insert b-gal gene

is interrupted by insert

Creating recombinant DNA with vectors Steps of molecular cloning

Fig 9.7 b

Identification of transformed bacterial cells with plasmids and inserts

Steps of molecular cloning

Fig 9.8

Plasmids and inserts are separated from bacteria and vectors by centrifugation, restriction digests and gel electrophoresis 2.3 Steps of molecular cloning: Purify cloned DNA

Fig 9.9 a, b

Video for DNA cloning



http://highered.mcgraw-hill.com/olc/dl/120078/micro10.swf

2.4 Libraries are collections of cloned fragments

How to compile a genomic library

 Complete genomic library – collection of clones that contains one copy of every sequence in the entire genome

Genomic equivalent – number of clones in a perfect library

Divide the length of the genome by the average size of inserts carried by the library’s vector

Researchers usually make libraries with four to five genomic equivalents for a 95% probability that each locus

is present at least once

cDNA libraries

Whole genomic libraries contain all DNA in a genome.

cDNA libraries carry information from the RNA

transcripts in a particular tissue

cDNA libraries contain only information from a gene’s

exons.

 Prepare total mRNA from a tissue

 Add reverse transcriptase and four deoxyribose

nucleotide triphosphates, and primers to initiate

synthesis

cDNA library construction

Fig 9.10

A comparison of genomic and cDNA libraries

Fig 9.11

Expression vectors produce large amounts of specific polypeptide

 Vector contains promotor and regulatory sequences.

 Vectors transformed into bacteria, yeast, or cultured mammalian cells

Fig 9.12 a

Screening an expression library

 Expression library – entire cDNA library

Probe library with fluorescently labeled antibody that binds to protein product of gene

Fig 9.12 b

3 Hybridization is used to identify similar DNA sequences

 Hybridization: DNA/DNA, DNA/RNA, RNA/RNA

 Prepare library

 Distribute library’s clones on petri dish

 Transfer clones to nitrocellulose disk

 Prepare probe

 Previous cloned DNA

 PCR fragment

 Oligonucleotide

 Screen library

 Expose probe to clones on nitrocellulose

 Determine location of matching clone

by autoradiography or fluorescence

Fig 9.13

Hybridization in DNA microarray

DNA microarray

Gel electrophoresis and hybridization to map DNA fragments

 Southern blot

 Cut whole genomic DNA with restriction enzyme

 Separate DNA fragments by electrophoresis

 Blot fragmented DNA to a filter

 Hybridize to DNA probe

 Observe matched bands by autoradiography or fluorescence

4 Polymerase chain reaction to rapidly isolate

DNA fragments

PCR (polymerase chain reaction) achieved

exponential accumulation of target DNA

 Based on previously determined DNA sequence,

develop short oligonucleotides (~ 20bp) complementary

to sequences flanking the target DNA

 Oligonucleotides act as primers to copy DNA similar to

DNA replication

Oligonucleotide primers begin copying DNA

Each cycle of replication doubles amount of target DNA Exponential amplification

PCR

Uses for PCR

Genetic mapping

Genotype detection

Analyze traces of partially degraded DNA

Evolutionary studies

 Compare homologous sequences from related

organisms

 Compare sequences from a variety of sources

 Studies of gene diversity

Diagnosis of infectious diseases

DNA Sequence Analysis

 All sequencing projects use same basic protocol

 Sequence determined approximately 800 bases at a time

 Maxim-Gilbert method

 Chemical cleavage of DNA at specific nucleotide types

 Sanger method

 Enzymatic extension of DNA to defined terminating base

 Sanger method most popular and efficient, particularly for automated methods

 Both techniques approximately 99.9% accurate

General Principals of Sanger Sequencing Method

Fig 9.17

Automated DNA sequencing

Fig 9.18 a

Automated DNA

 Output from an automated DNA sequencing reaction

Each lane displays the sequence obtained from

a separate DNA sample and primer

Automated DNA Sequencing

 Computer reads of the sequence complementary to the template strand from right to left (5’ – 3’ direction) Machine generates complementary strand Ambiguities are recorded as an “N” and can sometimes be resolved by a technician.

Fig 9.18 c

Sequencing long regions of DNA

 Primer walking

Sequence starting from both ends of cloned insert

New primers derived from sequence obtained in previous

round

 Shotgun sequencing

 Long DNA sequence chopped into many small fragments which are cloned individually

 Sequence of all small fragments determined

 Small fragments aligned

by computer to generate one long continuous sequence

Fig 9.19

Rapid sequencing

 Shotgun approach relies on redundancy

Must gather sequence information on 3-4 times the actual

number of base pairs from the original clone for full coverage

Sometimes must fill in gaps with primer walking

Must have many automated sequencers

Very fast if laboratory has enough equipment

 Primer walking

No redundancy required

No alignment necessary

Slower than shotgun sequencing because must make primers

after each round of sequence

Works well in laboratories without large number of automated

sequencers

Understanding the genes for hemoglobin: a comprehensive example

 Recombinant DNA technology used to isolate the a and b globin gene loci

Isolated RNA from red blood cell precursors

Produced cDNA libraries

Probed libraries for cDNA clones

Sequenced individual cDNA clones to identify a and b globin coding sequences

Used PCR to amplify sequences in many individuals with and without disease

Probed genomic DNA libraries to identify genomic clones and surrounding regions of globin genes

Used cDNA to probe Southern blots to determine number and location

of coding sequences within genomic locus

Sequenced entire chromosomal regions containing a and b globin genes

The genes encoding hemoglobin occur in two clusters on two

separate chromosomes

 The a-globin cluster contains three functional genes that spans

28 kb on chromosome 16

Fig 9.20 a

The genes encoding hemoglobin occur in two clusters on two separate chromosomes

 The b-globin cluster contains five functional genes and two pseudogenes spanning 50 kb on chromosome 11

Fig 9.20 b

The succession of genes in each cluster correlates with the sequence of expression

during development

a – globin cluster

  during the first five weeks of embryonic life

 Two a chains during fetal and adult life

b – globin cluster

  during first five weeks of embryonic life

 Two  chains during fetal life

 b and d within a few months of birth

 LCR associates with specialized DNA binding proteins at 5’ end of each gene cluster, bending chromosome back on itself to turn genes on and off in order.

 Example where adult b-globin genes are removed

 LCR cannot switch from activating fetal genes to activating adult genes.

 Fetal genes remain active in adult

Fig 9.20 c

A variety of mutations account for the diverse symptoms of globin-related

diseases

Two general classes of disorders

 Mutations alter amino acid sequence

Hemolytic anemias

e.g., sickle cell anemia – A-to-T substitution in sixth

codon of b-globin chain

 Mutations that reduce or eliminate production of one or

two globin polypeptides

thalassemia

Hemolytic anemias

Fig 9.21(a.1)

Sickle Cell Anemia

Fig 9.21 (a.2)

Thalassemias

Fig 9.21 (b.1)

Fig 9.21 (b.2)

Mutations in globin regulatory regions cause thalassemias.

 Mutations in the TATA box can eliminate transcription causing b thalassemia.

Fig 9.22 a

Mutations in globin regulatory regions cause

thalassemias.

 Mutation in the LCR can prevent expression of all a-globin genes

causing severe a-thalassemia

Fig 9.22 b

Evolution of the globin gene family

 Duplication of an ancestral gene followed by more duplications established the a and b-globin lineages

Fig 9.23