Question

DNA Technologies   


Imagine that you are a graduate student working in a research laboratory. You are interested in the human enzyme chymotrypsin. There is a lot known about this protein, but you have some specific questions about its mechanism. In order to study this protein further, you will need to amplify the chymotrypsin gene (you have only a small sample of the cDNA containing the gene), clone the gene into an expression plasmid, use bacterial cells to propagate the plasmid and express protein, and purify the protein. Use the attached information to plan out the first part of your experiments (amplification and cloning). Answer the following questions IN THE SPACE PROVIDED.


1. What restriction endonucleases will you use for cloning? Highlight their recognition sites on the attached template DNA sequence.





2. Design two DNA primers for use in amplifying the gene. Give their sequences below (5' to 3') and label where they will bind on the attached template DNA sequence.







3. Outline the process of amplifying the gene (using PCR), then cloning, including all reaction components for each process.

PCR amplification  

Steps reaction components

Cloning

Steps reaction components

Type II Restriction Endonucleases
Enzyme Source Recognition Sequence EcoRI Escherichia coli 5'G│AATT C 3'C TTAA│G

BamHI Bacillus amyloliquefaciens 5'G│GATC C \

3'C CTAG│G

HindIII Haemophilus influenzae 5'A│AGCT T

3'T TCGA│A

NotI Nocardia otitidis 5'GC│GGCC GC

3'CG CCGG│CG

SmaI* Serratia marcescens 5'CCC│GGG

3'GGG│CCC

HaeIII* Haemophilus aegyptius 5'GG│CC

3'CC│GG

AluI* Arthrobacter luteus 5'AG│CT

3'TC│GA

EcoRV* Escherichia coli 5'GAT│ATC

3'CTA│TAG

KpnI Klebsiella pneumoniae 5'G GTAC│C

3'C│CATG G

PstI Providencia stuartii 5'C TGCA│G

3'G│ACGT C

SalI Streptomyces albus 5'G│TCGA C

3'C AGCT│G

SpeI Sphaerotilus natans 5'A│CTAG T

3'T GATC│A

SphI Streptomyces phaeochromogenes 5'G CATG│C

3'C│GTAC G

XbaI Xanthomonas badrii 5'T│CTAG A

3'A GATC│T
Key: * = blunt ends

DNA Sequence of Chymotrypsin in Context

GACCTATTAG GAATAAACAG GATACCATTG GCAAGCTTAC GTACACGACT TGATTGACAC TTACCCAGTA GGGGACGAAT GGATCCATCG AAGGCTGCAG GGATGAATTC TTTCACTATG TGTGGGGTGC CCAGCTTCCC GCCCAACCTA TCCGCCCGAG TGGTGGGAGG AGAGGATGCC CGGCCCCACA GCTGGCCCTG GCAGATCTCC CTCCAGTACC TCAAGGACGA CACGTGGAGG CATACGTGTG GCGGGACTTT GATTGCTAGC AACTTCGTCC TCACTGCCGC CCACTGCATC AGCAACACCT GGACCTACCG TGTGGCCGTG GGAAAGAACA ACCTGGAGGT GGAAGACGAA GAAGGATCCC TGTTTGTGGG TGTGGACACC ATCCACGTCC ACAAGAGATG GAATGCCCTC CTGTTGCGCA ATGATATTGC CCTCATCAAG CTTGCAGAGC ATGTGGAGCT GAGTGACACC ATCCAGGTGG CCTGCCTGCC AGAGAAGGAC TCCCTGCTCC CCAAGGACTA CCCCTGCTAT GTCACCGGCT GGGGGCGCCT CTGGACCAAC GGCCCCATTG CTGATAAGCT GCAGCAGGGC CTGCAGCCCG TGGTGGATCA CGCCACGTGC TCCAGGATTG ACTGGTGGGG CTTCAGGGTG AAGAAAACCA TGGTGTGCGC TGGGGGCGAT GGCGTTATCT CAGCCTGCAA TGGGGACTCC GGTGGCCCAC TGAACTGCCA GTTGGAGAAC GGTTCCTGGG AGGTGTTTGG CATCGTCAGC TTTGGCTCCC GGCGGGGCTG CAACACCCGC AAGAAGCCGG TAGTCTACAC CCGGGTGTCC GCCTACATCG ACTGGATCAA CGAGAAAATG CAGCTGTGAT TTGTTGCTGG GAGCGGCGGC AGCGAGTAAT AGTAGTACGG AAGTGCGGCC GCTAGACTGA GATATCGGCC TATCCGATAT CGATAGGGTC ATTAAGAACG GCTCGATAG

Answer 1

1) Restriction endonucleases:

The enzymes that cleave DNA molecules at specific nucleotide sequences called restriction sites
"molecular scissors" - discovered in bacteria, isolated by Nathans and Smith in 1970
- each bacterial nuclease recognizes a sequence of 4-8 nucleotides in the DNA
- 3000 enzymes have been ID'd, ~200 w/ unique properties
- names are based upon bacterial genus, species, strain, and order of discovery

In the natural host, their function is to recognize foreign DNA sequences and cut it into pieces for further degradation by host exonucleases.It restricts what type of DNA can exist in the host.

Cell-based DNA cloning is the cutting a piece of DNA from one organism and inserting it into a vector where it can be replicated by a host organism. cell/organism cloning using nuclear DNA from one organism to create a second organism w/ the same nuclear DNA

uses of cloning

- determine the sequence of recombinant DNA
- generate a probe for hybridization
- recombinant expression of the encoded protein and mutagenesis
- regenerative medicine - transplant

The Titanium 454 machine and the Solexa/Illumina machine, both use two key changes in approach. First, there is no cloning. Fragments of DNA are amplified and then used for sequencing while linked to a surface (either a flow cell or a bead), saving all the work of cloning and subcloning the DNA in the sample. Second, CCD cameras are used to observe synthesis as it occurs, recording either a color-coded flash of light for incorporation of a specific base (all four present), or a flash of light following incorporation of a specific base (only one at a time present). This eliminates the need for size- separation of sequencing products - no gel electrophoresis is needed - and allows sequencing of thousands of DNA fragments at the same time.
Several kinds of experiments are made possible by next-generation sequencing. Once we have a good version of a genome sequence, we can "resequence" DNA from individuals of that species, allowing analysis of population genetics, analysis of the genomes of cancer cells, etc. It also allows "metagenomics" - sequencing and comparing pools of DNA in a sample taken from the environment. Another big use is "ChIP" experiments - chromatin immunoprecipitation. Here we cross-link the chromosomal proteins to DNA, shear the cross-linked chromatin to generate small fragments, and selectively precipitate that fragments that have a particular protein (such as RNA polymerase) bound by using specific antibodies; sequencing the DNA fragments from the bound material gives us a map of the sites in the genome where RNA polymerase is bound.