Structural tics Reading Dna Fingerprints of Excised Vectors

20
STRUCTURAL BIOINFORMATICS, PROTEOMICS AND MICROARRAYS Submitted by Srinivas Mukund Vadrev M.S.C Analytical Biosciences and Drug Design EXPERIMENT 1

Transcript of Structural tics Reading Dna Fingerprints of Excised Vectors

Page 1: Structural tics Reading Dna Fingerprints of Excised Vectors

STRUCTURAL BIOINFORMATICS, PROTEOMICS AND MICROARRAYS

Submitted by

Srinivas Mukund Vadrev

M.S.C Analytical Biosciences and Drug Design

EXPERIMENT 1

Page 2: Structural tics Reading Dna Fingerprints of Excised Vectors

Identification of mouse NEIL cDNA inserts in pGEM-T PCR cloning vectors by Restriction Endonuclease Mapping.

AIM:

1. To use Restriction Endonucleases and carry out Restriction digestion of pGEM T and pGEM T Easy vectors containing NEIL cDNA (NEIL1 , NEIL2 and NEIL3) inserts and to identify the vector and .

2. To record the results for further analysis using restriction endonuclease mapping and molecular databases.

INTRODUCTION

Restriction Endonucleases are a class of enzymes involved in a genetic restriction modification system.These restriction enzymes are able recognize specific nucleotide sequences and cleave them at specific sites.DNA glycolases are a kind of restriction enzymes.DNA can be damaged by a variety of factors. For instance ionizing radiation produces a range of damage types in cellular DNA.Base excision repair is one of the mechanisms of DNA repair. This repair mechanism is catalyzed by a glass of enzymes called DNA glycolases.The first step that occurs is the excision of of the base lesion. DNA glycolases cleaves the N-glycosidic bonds of the faulty base to for an Abasic site or AP site,These sites are empty sites containing neither a purnine or pyrimidine base.These AP sites are cleaved by an 5′ AP endonuclease type enzyme to leave a 5′ dRp terminus (5' deoxyribosephosphate).This is removed by the action of DNA polymerase producing a single nucleotide gap.Then enzyme AP lyases by beta elimination generate a 3′ OH end from its phosphatise domain and a 5′ OH from its kinase domain.DNA polymerase β then typically inserts a single nucleotide.The 5′ OH and 3′ OH are acted upon by flap endonucleases and get excised.Finally DNA ligases catalyzes and seals the nick closed.

Procedure :

Step 1:

Page 3: Structural tics Reading Dna Fingerprints of Excised Vectors

The first step was to prepare 3 digests. The first Digest is an EcoR1 digest of 3 DNA samples labelled A1,B1 and C1.This is to help locate which vector the NEIL cDNA has been introduced into.

The second digest is a double digest containing NdeI and Hind III restriction enzymes. Labelled A2,B2 and C2.These enzymes will cleave the cDNA insert and reveal which sample contains NEIL1,NEIL2 and NEIL3.

The third digest contains an extra restriction enzyme Xmn I.Each NEIL cDNA insert contains a unique Xmn I site.Hence each NEIL cDNA fragment cut is obtained of the same size.

DIGEST 1

3 x 0.5 ml microcentrifuge tubes were obtained and were labelled as A1, B1 and C1.DNA samples A, B and C were transferred to tubes A1, B1 and C1 respectively.The reagents for the preparation of the restriction were prepared (including DNA samples) according to the following formula.

DNA sample 5.0 micro Litres

10x NEB buffer 2 2.0 micro Litres

ECoRI 5.0 micro Litres

dH2O 8.0 micro Litres

----------------------------------------------------------

TOTAL 20 micro Litres

--------------------------------------------------------------

Using a P20 pipette, reagents for restriction digest along with corresponding DNA samples (A , B and C) were transferred to respective microcentifuge tubes. The mixture in the tubes was homogenized by vortexing briefly for about 1-2 seconds allowing the centrifuge to reach a speed of ~5000rpm.The tubes were then incubated for 1h at 37*C in a water bath.

DIGEST 2

Page 4: Structural tics Reading Dna Fingerprints of Excised Vectors

As similar to procedure in DIGEST 1, 3 x 0.5 ml microcentrifuge tubes were obtained and labelled as A2, B2 and C2.Corresponding DNA samples for the 3 tubes with restriction digest mixture were transferred. They were then centrifuged till the centrifuge reaches ~5000rpm and then incubated for 1 hour in a water bath at 37*C.

DNA sample 5.0 micro Litres

10x NEB buffer 2 2.0 micro Litres

NdeI 5.0 micro Litres

HindIII 5.0 micro Litres

dH2O 3.0 micro Litres

----------------------------------------------------------

TOTAL 20 micro Litres

--------------------------------------------------------------

DIGEST 3

As similar to procedure in DIGEST 1, 3 x 0.5 ml microcentrifuge tubes were obtained and labelled as A3, B3 and C3.Corresponding DNA samples for the 3 tubes with restriction digest mixture were transferred. They were then centrifuged till the centrifuge reaches ~5000rpm and then incubated for 1 hour in a water bath at 37*C.

DNA sample 5.0 micro Litres

10x NEB buffer 2 2.0 micro Litres

10x BSA 2.0 micro Litres

XmnI 5.0 micro Litres

dH2O 6.0 micro Litres

----------------------------------------------------------

TOTAL 20 micro Litres

--------------------------------------------------------------

Step 2:

Preparation of 0.8% agarose gel

Page 5: Structural tics Reading Dna Fingerprints of Excised Vectors

475ml of deionised water was taken in a 500ml measuring cylinder and 25ml of 10x TBE buffer solution was added to make the volume upto 500ml.The mixture was then homogenised by stretching a parafilm over the mouth of the cylinder and then inverting 2-3 times.

0.24g of agarose was weighed and transferred into a 250ml conical flask and 30ml of 0.5X TBE buffer was added to it. The flask was plugged with a piece of cotton wool and heated in short bursts (30 seconds) by microwaving on medium setting till the mixture boils and agarose has fully dissolved.

The agarose solution was allowed to cool to 55*C and 30 micro Litres of Gel Red was added. The Flask was shaken and swirled to ensure uniform mixing.The gel was then poured into the gel tray and a comb was inserted to create wells and then left aside to set.

Step3

The tubes were removed after 1h from the water bath and pulse spun to remove any sort of condensation that might have formed on the lid of the tube.

The comb was removed and 3.0 micro Litres of loading buffer was added to each sample and mixed. To WELL 1, 10micro Litres of DNA size marker was added. The prepared samples were then added in order of A1,B1 & C1; A2,B2 & C2 and A3,B3 and C3.

Step4:

With the wells facing the negative terminal of the gel electrophoresis apparatus and lid in place, a voltage of 100v was delivered. Making sure current is flowing, it was allowed to run for 45-50 min.

Once the time interval has lapsed, the gel was placed on a UV-transilluminator to visualise DNA bands. A picture was then taken for analysis.

RESULT

A1 B1 C1 A2 B2 C2 A3 B3 C3

Page 6: Structural tics Reading Dna Fingerprints of Excised Vectors

OBTAINED RESULT

CONTROL

In A1,B1 and C1, 2 bands are observed in C1 while only 1 band is observed in A1 and B1.Hence EcoR1 site is present in C1.Thus it can be concluded that A1 and B1 are pGEM-T vectors while C1 contains pGEM-T vector.

In digest 2, the fragment separated at A2 is the longest one separated.Hence the fragment separated at A2 can be NEIL3 which is the longest in base pairs.Well C2 is NEIL1 because it is the second longest.The third at B2 separated the least towards the negative terminal and thus it is NEIL2.In digest 3 , specific sized fragments of NEIL1,NEIL2 and NEIL3 were obtained.Their sizes were calculated as follows

Calculation

Band 1

NEIL I size= 707

size of vector = 3000

XmnI cuts at base pair = 1994

remaining base pair = 3000-1994

Page 7: Structural tics Reading Dna Fingerprints of Excised Vectors

= 1006

3’OH end 1006+46= 1052

NEIL I + 1052 = 707+ 1052= 1759

Similarly the calculation for the others was carried out.

EXPERIMENT 2

Analysis of DNA Sequences using Restriction Mapping and Molecular Databases.

AIM:

1. To investigate the results obtained above further using restriction endonuclease mapping and molecular databases.

2. To determine which NEIL homolog is associated with which vector.

3. To obtain nucleotide and protein sequence of mouse NEIL homolougs.

4. To determine the identity of PCR cloning vector for each DNA sample.

5. To obtain further information on NEIL homologues.

Page 8: Structural tics Reading Dna Fingerprints of Excised Vectors

INTRODUCTION:

Restriction mapping is a method of obtaining structure related information of a given DNA sample such as length of the fragment in base pairs; location of restriction enzyme sites etc.This can be obtained as preliminary information to carry out further investigations. It involves the digestion of given DNA sample using restriction enzymes and then analysing using gel electrophoresis. The distance between the restriction site can be estimated from looking at the patterns and arrangement of fragments. Restriction mapping can also be used to analyse the sequence of large sequences of DNA by fragmentation. Gel electrophoresis is carried out and sizes of the fragments is determined by using Restriction mapping software such as

VECTOR NTI (http://www.invitrogen.com/)

NEBcutter(http://tools.neb.com/NEBcutter2/index.php

The NCBI or National Centre for Biotechnology information is the largest and most comprehensive compilation of publicly accessible DNA sequences, biomedical and genomic information and molecular biology information.

Procedure

To carry out the restriction mapping on the pGEM-NEIL cDNA samples, mRNA nucleotide sequences

for NEIL1,NEIL2 and NEIL3 have to be obtained.These mRNA sequences can be obtained from NCBI’s

molecular gene database.

The following url was typed into the browser and navigated to.

http://www.ncbi.nlm.nih.gov/

Page 9: Structural tics Reading Dna Fingerprints of Excised Vectors

For search query, NEIL1 was entered and was searched for under ‘Gene’.The following results were

displayed.

Page 10: Structural tics Reading Dna Fingerprints of Excised Vectors

In the search results, the third entry is for NEIL1 [Mus musculus].The result was opened by clicking on

it.

NEIL1 translated sequence

1 atgccagagg gcccagagct gcacctggcc agccactttg tgaatgagac atgtaagggg

61 ctggtatttg gtgggtgtgt ggagaagtcc tctgtcagcc ggaacccgga ggtgcccttt

121 gagagcagtg cctaccacat ctcagcttta gcccgaggca aggagctgcg cttgacattg

181 agccccctgc ctgggtccca gccccctcag aagccactgt cccttgtctt ccgctttggg

241 atgtcaggat ccttccagct ggtacccgca gaggcactgc cccgccacgc ccatctacgt

301 ttttacacag ccccacctgc tccccggctt gccctttgct tcgtagacat ccgtcgcttt

361 ggccactggg atcctggggg tgaatggcaa ccaggccgtg gaccctgtgt cttgctggag

421 tatgaacggt tcagagagaa cgtacttcgg aacctatcag acaaagcctt tgaccggccc

Page 11: Structural tics Reading Dna Fingerprints of Excised Vectors

481 atctgtgagg ccttgttgga ccagaggttc ttcaatggca ttggcaacta tctgcgggca

541 gagatcctgt accggctgaa gatccctcct tttgagaagg ctcgtacagt tctagaggcc

601 ctgcaacagt gccggccgag cccagagctg accctgagcc agaagatcaa ggccaaacta

661 cagaacccag acctgctgga actgtgtcac ttggtgccca aggaagtggt tcagctgggg

721 ggcaaaggct atgggccaga gcgtggagag gaggattttg ctgcctttcg agcctggctt

781 cggtgctatg gtgtgccagg catgagctcc ctgcgagacc ggcatggccg taccatctgg

841 ttccagggtg atcccggacc cttggcaccc aaagggggca gatcccaaaa aaagaagtca

901 caggagacac agctgggggc tgaggacagg aaagaggacc ttccactttc aagcaagtcc

961 gtttccagga tgcggagggc caggaagcac cctcctaaga gaatagctca acagtctgaa

1021 ggggccggcc tccaacaaaa ccaggaaacc cctacagctc ctgagaaagg gaagaggagg

1081 gggcaacggg cgagcacagg ccaccgcaga cgcccaaaga ctatacctga cacccgaccc

1141 agggaggctg gggagagttc agcttcatag

Restriction mapping was carried out by navigating to web mapper hosted by Harvard Medical school at:

<http://pga.mgh.harvard.edu/web_apps/web_map/start >

Page 12: Structural tics Reading Dna Fingerprints of Excised Vectors

The sequence for NEIL1 was copied into the box and was submitted.

Page 13: Structural tics Reading Dna Fingerprints of Excised Vectors

Xmn I site for NEIL1:

Xmn1 nnnGAANN NNTTCnnn nnnCTTNN NNAAGnnn

Cuts at: 0 707 1179 Size: 707 472 Fragments sorted by size: 707 472

Similarly the CDS for NEIL2 and NEIL3 was found out

NEIL2 translated sequence

1 atgccagaag ggccatctgt gaggaagttt caccatcttg tctccccctt tgtgggccag

61 aaggtggtca agacgggggg cagcagtaag aagctccacc ctgccgcctt tcagtctctg

121 tggctccagg atgctcaggt gcatggaaaa aaattattcc ttcggtttga tccagatgag

181 gagatggagc cactcaacag cagcccacag cctatacagg gaatgtggca gaaagaggct

241 gtggaccgag agctggcctt gggtcccagt gctcaggaac cctctgcagg tccctctgga

301 tctggggagc ctgttcccag tagatctgct gaaacatata atcttgggaa gatcccctca

361 gcagatgccc agaggtggct ggaggtcagg tttggtttat ttggcagtat ctgggtgaat

421 gacttctcca gagcaaagaa agctaacaaa aaaggtgact ggagagaccc agtgcccagg

481 ctggtactcc attttagtgg tggtggcttc ctggtatttt ataactgcca gatgtcatgg

541 agccctcccc cagtgattga gcccacctgt gacatattgt ctgaaaagtt ccatcgagga

601 caagccttgg aagctctaag ccaggctcag cctgtgtgct acacactctt ggaccagaga

661 tacttctcag gattagggaa catcataaag aatgaagcct tgtacagagc aaggatccat

Page 14: Structural tics Reading Dna Fingerprints of Excised Vectors

721 cccctctctc tcggttcatg cctgagttct tcctctcggg aggccctcgt ggatcacgtg

781 gtggagttca gtaaggactg gcttcgggac aaattccaag gcaaggaacg gcacacacag

841 atctaccaga aggaacaatg tccttctggt caccaggtca tgaaggagac atttgggccc

901 ccagatgggc tccagaggct cacctggtgg tgccctcaat gccagcccca gctgtcctcc

961 aaggggcccc agaatctccc gtcctcctaa

Xmn I site for NEIL2:

Xmn1 nnnGAANN NNTTCnnn nnnCTTNN NNAAGnnn

Cuts at: 0 421 990 Size: 421 569 Fragments sorted by size: 569 421

NEIL3 translated sequence

1 atggtggaag ggccagggtg tacactgaat ggagagaaga tccgggcacg agtgctaccg

61 gggcaggcgg tgactggtgt gcggggcaca gctctgcaga gcctcctggg ccctgccatg

121 tcccctgccg cctccctggc tgatgtcgct acctcggctg ctccaatgaa tgctaaggat

181 tctggctgga aactcttgag actgtttaat ggatatgttt acagtggcgt ggaaacctta

241 gggaaggagc tgtttatgta ctttggcccc agagctttac ggattcattt cggaatgaaa

301 ggctccatct tgattaaccc acgggagggt gagaacagag ctggggcttc tccagccttg

361 gcggtgcagc tcaccagaga cttgatctgc ttctatgact cttcagtaga actcagaaat

421 tcggtggaaa gccaacagag agtcagagtg atggaagagt tggatatatg ttcaccaaag

481 ttcagtttct caagagcaga gagtgaagtg aaaaagcagg gagatcggat gctgtgtgat

541 gtgttgctgg atcagagggt gttgcctggc gtgggcaaca tcatcaaaaa tgaagcactc

Page 15: Structural tics Reading Dna Fingerprints of Excised Vectors

601 tttgacagtg gtcttcatcc ggctgttaag gtgtgtcaac tatcagacaa acaggcctgt

661 caccttgtga agatgactcg ggatttcagc attctcttct acaggtgctg taaagcagga

721 tctgccattt ctaaacactg taaggtttac aagcgtccta actgtgatca gtgccacagc

781 aaaattactg tgtgtcgctt tggggagaac agcaggatga catatttctg tcctcactgt

841 cagaaagaaa accctcaatg tgttcaagta tgccagctgc caacaagaaa cactgaaatc

901 agctggactc ctaggggaga ggattgcttt acggactcag tggctcggaa gtctgaagag

961 cagtggtcct gtgtggtttg tactctcata aacagaccct cggctaaggc ctgtgatgct

1021 tgcttgacca caaggcctct ggattcagtg ctcaagaata gagaaaattc cattgccttc

1081 aacaacttag tgaagtaccc ttgtaataac tttgagaaca cacacactga agtaaagatc

1141 aacaggaaaa ctgcgtttgg aaatacgacc cttgttttga ctgatttaag caataaatcc

1201 agtgctttgg ccagaaagaa aagagcaaac cacacaatag atggggaatc tcaaatgttt

1261 ctccctacag acataggttt tagtgattca cagcacccct ccaaagaagg aataaactat

1321 ataactcaac catccaataa ggtaaacata tcacctacag tttgtgccca gtctaaatta

1381 tttagttcag cacataaaaa attcaaacca gctcacactt ctgcaacaga acttaaaagt

1441 tacaactctg gactttctaa cagtgaactc caaaccaata ggacacgtgg ccatcattcc

1501 aaaagtgatg gcagccctct gtgcaagatg caccaccgcc gttgtgttct ccgagttgtg

1561 aggaaagatg gagaaaacaa ggggaggcag ttttatgcct gttctctgcc gagaggagca

1621 cagtgcggat tttttgaatg ggcagacctg tccttcccgt tctgcagaca tggcaagcgc

1681 tccattatga aaactgtgct gaagattgga cctaataatg ggaagaattt ttttgtatgt

1741 cctttggaga aaaaaaagca gtgtaatttt ttccagtggg ccgaaaatgg accaggaatg

1801 gaaattgttc caggatgcta a

Xmn I site for NEIL3:

Xmn1 nnnGAANN NNTTCnnn nnnCTTNN NNAAGnnn

Cuts at: 0 1805 1821 Size: 1805 16 Fragments sorted by size:

Page 16: Structural tics Reading Dna Fingerprints of Excised Vectors

1805 16

BLAST:

Its is Basic Local Alignment Search Tool.To obtain more information on the homology between NEIL proteins, BLAST has to be run.

http://blast.ncbi.nlm.nih.gov/Blast.cgi

To check if there is any homology in nucleotide sequence of bacterial with NEIL proteins,a search was conducted for ‘nei and e cole’.The following is the result.

Page 17: Structural tics Reading Dna Fingerprints of Excised Vectors

Clicking NC_000913.2 and choosing the FASTA format, the following DNA sequence of nei gene was obtained....

FASTA SEQUENCE: NEI

>gi|16128689|ref|NP_415242.1| endonuclease VIII/ 5-formyluracil/5-hydroxymethyluracil DNA glycosylase [Escherichia coli str. K-12 substr. MG1655]MPEGPEIRRAADNLEAAIKGKPLTDVWFAFPQLKPYQSQLIGQHVTHVETRGKALLTHFSNDLTLYSHNQLYGVWRVVDTGEEPQTTRVLRVKLQTADKTILLYSASDIEMLTPEQLTTHPFLQRVGPDVLDPNLTPEVVKERLLSPRFRNRQFAGLLLDQAFLAGLGNYLRVEILWQVGLTGNHKAKDLNAAQLDALAHALLEIPRFSYATRGQVDENKHHGALFRFKVFHRDGEPCERCGSIIEKTTLSSRPFYWCPGCQH

The sequence was then pasted into BLASTN and was searched.

BLAST SEARCHExpect parameter 0.01

Page 18: Structural tics Reading Dna Fingerprints of Excised Vectors

Expect parameter 0.01The “super families” box in the graphical summary reveals us more information regarding conserved domain.

There is homology between nei in E.coli.

Page 19: Structural tics Reading Dna Fingerprints of Excised Vectors

REFERENCES

1. Fortini and Dogliotti(2006) Base damage and single-strand break repair: Mechanisms and functional significance of short- and long-patch repair subpathways. 2006.10.008 [online]<http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6X17-4MFK44B-1&_user=899537&_coverDate=04/01/2007&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000047642&_version=1&_urlVersion=0&_userid=899537&md5=051b628e0a4a6d6f0a22a1b147220f83&searchtype=a>

[Accessed: 10th November 2010]

2. Richard J. Roberts and Kenneth; Restriction Endonuclease ;Murray1976, Vol. 4, No. 2 , Pages 123-164 (doi:10.3109/10409237609105456)http://informahealthcare.com/doi/abs/10.3109/10409237609105456

[Accessed: 10th November 2010]

3.Ross A. Lippert; Brute force and digest problems;

<http://www-math.mit.edu/~lippert/18.417/lectures/02_PartialDigest/>[Accessed: 10th November 2010]

[Accessed: 11th November 2010]

4.Restriction Mapping<http://faculty.plattsburgh.edu/donald.slish/Restmap.html>

<http://www.virology.net/Articles/biotechniquessites1.html>

[Accessed: 10th November 2010]

Page 20: Structural tics Reading Dna Fingerprints of Excised Vectors