Molecular Techniques

Question 1

What do Southern and Northern Blot analysis of NA detect?
What do they BOTH START WITH?

Answer 1

Southern & Northern blot analysis of NA detects a MOLECULE
within a mixture

• starts with electrophoresis

Question 2

Southern & Northern blot analysis Technique 10

Answer 2

1. 32p-labeled size markers filled with RNA or DNA
2. Electrophoresis
   - migration from - to +
3. In Gel; with paper towels, salt solution, membrane, sponge
   SOLUTION PASSES THROUGH GEL AND MEMBRANE TO PAPER TOWELS
4. Gel+Filter
   - DNA transferred to MEMBRANE
5. Hybridise with unique nucleic acid probe
6. Filter in “seal-a-meal” bag.
7. Remove unbound probe
8. Probe hybridised to complementary sequence
9. Expose X-ray film to membrane.
10. Autoradiogram

Question 3

Northern vs Southern vs Western…

Answer 3

Procedure is TERMED SOUTHERN blotting when DNA is TRANSFERRED (named after EDWIN SOUTHERN)
-NA probe used

NORTHERN BLOTTING when RNA is TRANSFERRED
-NA probe

WESTERN blotting transfer of PROTEIN to a MEMBRANE, USE OF ANTIBODIES NOT A NA PROBE

Question 4

What does DNA sequencing Determine?

Answer 4

Determines the sequence of bases in DNA

Question 5

In mid 1970s, FREDERICK SANGER created the dideoxysequencing method…EXPLAIN IT 4

Answer 5

1. Based on the elongation of DNA by DNA polymerase (REPLICATION)
2. FRAGMENT to be SEQUENCED is USED as TEMPLATE
3. REPLICATION is sometimes TERMINATED when a SPECIFIC BASE is ENCOUNTERED
4. Dideoxyribonucleoside triphosphate (ddNTP) lacks a 3’-OH group, which terminates DNA synthesis.

Question 6

Understanding ddNTPS…4

Answer 6

1. ddNTPs are ALMOST identical to dNTPs but THEY LACK 3;-OH group.
2. if a ddNTP is INCORPORATED into DNA strand, NO MORE NUCLEOTIDES can be ADDED.
3. No free 3’-OH to form phosphodiester bond with incoming nucleotide.
4. ddNTPs terminates synthesis.

Question 7

Describe the STEPS (10)
Sanger DNA sequencing method….

a technique used to determine the sequence of nucleotides in a DNA molecule.

Answer 7

1. Each of four reactions contains: single-stranded target DNA to be sequenced…
2. …a PRIMER
3. ..all 4 deoxyribonucleoside triphosphate, DNA polymerase…
4. …and one type of dideoxyribonucleoside triphosphate (ddNTP).
5. Nucleotides are added to the 3’ end of the primer, with the target DNA being used as a template.
6. When a dideoxynucleotide is incorporates into growing chain, synthesis terminates because the dideoxynucleotide lacks a 3’ OH.
7. Synthesis terminates at different positions on different strands, which generates a set of DNA fragments of various lengths, each ending in a dideoxynucleotide with the same Base.
8. The fragments produced in each reaction are separated by gel electrophoresis.
9. the sequence can be read directly from the bands that appear on the autoradiograph of the gel, starting from the bottom.
10. The sequence obtained is the complement of the original template strand.

Question 8

The steps (7) describe a variation of the Sanger DNA sequencing method, known as FLUORESCENT DYE TERMINATOR SEQUENCE.

Answer 8

1. A single-stranded DNA fragment whose base sequence is to be determined (the template) is isolated.
2. Each of the 4 ddNTPs is tagged with a different fluorescent dye, and the Sanger sequencing reaction is carried out.
3. The fragments that end in the same base have the same coloured dye attached.
4. The products are DENATURED, and the DNA fragments produced by the 4 reactions are mixed and loaded into a single well on an electrophoresis gel. The fragments migrate through the gel according to size…

5….and the fluorescent dye on the DNA is detected by a laser beam.

6. each fragment appears as a peak on the computer print out, the colour of the peak indicates which base is present.
7. the sequence information is read directly into the computer, which converts it into the complementary target sequence.

Question 9

The meaning of the Genomic Sequence:

DRAW AND LABEL IT PG. 10.

Answer 9

1. Regulatory proetin binds DNA.
2. RNA polymerase binds DNA
3. ribosome binds mRNA
4. tRNAs bind each codon in mRNA.
5. Spliceosome binds primary RNA transcript.
6. Translation termination protein binds mRNA.
7. Poly(A) polymerase binds primary RNA transcript.
8. Transcription regulatory element
9. Promoter
10. 5’ UTR
11. Translation initation site
12. Codons
13. Intron
14. Codons
15. Exon
16. 3’ Splice site
17. Codons
18. Translation termination site
19. 3’ UTR
20. Polyadenylation site

Question 10

How is information encoded into DNA? =4

Answer 10

1. SUM OF ALL GENE PRODUCTS - PROTEINS AND RNAs.
2. More complex than that - another view: GENOME encodes a SERIES of DOCKING sites FOR DIFFERENT PROTEINS AND RNAs.
3. Complex set of networks determine WHERE, WHEN AT WHAT LEVEL GENE WILL BE EXPRESSED.
4. Therefore, the information “ENCODED” in genome=
   SUM OF ALL SEQUENCES THAT CODE PROTEIN and RNAs, plus the DOCKING SITES THAT PERMIT THEIR PROPER ACTION IN TIME AND TISSUE.

Question 11

in addition to protein encoding sequences, GENOME CONTAINS ADDITIONAL INFORMATION and ITS NOT CLEAR WHAT ALL FO NUCLEOTIDE SEQUENCE MEANS

UNCERTAINTY DUE TO: 2

Answer 11

1. Docking (TARGET) sequences of many DNA binding proteins are UNKOWN.
2. ALTERNATIVE SPLICING COMPLICATES “ORF” finding some SEQUENCES have MULTIPLE USES.

Bioinformatics and functional genomics attempt to decipher genome.

Question 12

Bioinformatics: What is it What does is Show and how? 4

Answer 12

1. Uses available information (much of it available on the.
   Web) to predict function of sequences
2. cDNA.evidence
   - motifs, e.g..start codons, ORFs
   - expressed sequence tags (ESTs), from reverse transcribed mRNA
3. mRNA & ORF structure
   - gene &. intron finding programs

4.Polypeptide similarity
- at evidence at level of >35% sequence identity, polypeptides likely have common function

• often identified by BLASTp search Codon bias information

Question 13

Identifying Genes from
”Novel” Sequence
(predicting function from sequence) -OPEN READING FRAMES

WHAT IS IT? WHAT IS A CANDIDATE GENE?

Answer 13

1. A stretch of nucleotides beginning with ATG
   (initiation codon) & ending with an in frame stop
   codon is called an open reading frame (ORF).
2. An ORF represents a CANDIDATE GENE that
   encodes a protein.
   Based on the genetic code,
   one can determine the amino acid sequence of
   the protein encoded by he ORF.

Question 14

cDNA evidence - ESTs

Answer 14

1. cDNA: extremely valuable in identifying exons (DNA copies of mRNA)
   - Alignment with genomic sequence delineates exons
   - in cDNA ORF continuous through to stop codon
   - can assist in identifying correct reading frame.
2. ESTs: expressed sequences tags - in many genes only the 5’ or 3’ ends of the cDNA sequenced - can be aligned with genomic DNA, determine transcript ends.

Question 15

Understanding Polypeptide similarity evidence:

Answer 15

Candidate ORFs can be given credibility by comparing with all other genes.

Submit candidate sequences to public databases - BLAST search.

Since organisms share common ancestors, gene sequences are generally similar.

A “REAL” gene will likely be found in other Organisms.

Question 16

Classifying Genes: what happens after genomic sequencing?

Answer 16

After the genomic sequence is annotates, homology searches are performed to try to assign a function to each ORF.

About half the ORFs in bacterial genomes have no Known function.

Question 17

PUTTING IT ALL TOGETHER - DNA SEQUENCING..

Answer 17

Putting it all together:
- different sources of information are combined to create the
best possible (most likely) mRNA & ORF predictions

e.g. structure of gene may be inferred from evidence of
protein similarity within region of genomic DNA

• docking site predictions &’codon bias as supporting evidence

Question 18

Understanding Functional genomics: 4

Answer 18

1. Study of expression and interaction of gene products
2. Geneticists have been studying expression and interactions of gene products for 50+ years
3. Has been small scale, pone gene at a time approach.
4. Expand studies to a global level - look at most or all gene products simultaneously.

Question 19

Understanding the CHALLENGE of FUNCTIONAL genomics = 4

Answer 19

1. Capacity for collecting data has surpassed the data analysis techniques …still expanding
2. Converting data (information) into knowledge is a bottleneck
3. Currently requires expertise and a labour-intensive “hands -on” approach
4. Ultimate goal is to provide more automation to the process of knowledge discovery.

Question 20

Genomics spawned “omics era” = new vocab and techniques

Answer 20

Genomics spawned “omics era” = new vocab and techniques

Question 21

Understanding
Transcriptome:

Answer 21

ALL RNA TRANSCRIPTS

• sequence and expression patterns of all transcripts (where, when, how much)
• • may be monitored by used of DNA chips (microarrays)

Question 22

Understanding PROTEOME:

Answer 22

ALL ENCODED PROTEINS

• sequence and expression patterns of all proteins
• complicated by alternative splicing

Question 23

understanding INTERACTOME:

Answer 23

ALL INTERACTIONS BETWEEN ALL CATEGORIES OF MOLECULES

• between proteins/DNA segments, proteins/RNA and protein/protein
• detected by 2-hybrid system and related procedures

Question 24

Understanding PHENOME

Answer 24

SET OF PHENOTYPES PRODUCED IN RESPONSE TO GENETIC MUTATION AND ENVIRONMENTAL INFLUENCES

• phenotype of each gene knockout (for each gene in genome)

Question 25

What is Recombinant DNA technology

Answer 25

A set of techniques for LOCATING, ALTERING and STUDYING DNA SEGMENTS. RECOMBINANT = combine DNA from 2 distinct sources (e.g. genes from 2 different bacteria; human gene into a viral chromosome)

Question 26

2. The molecular genetics revolution- EXPLAIN HOW RECOMBINANT DNA

Answer 26

Drastically altered way genes are studied ie. not by phenotype Our understanding of genetic processes from these techniques Biotechnology industry grown up using these techniques to develop new products ie drugs, hormones, enzymes etc.

Question 27

ISOLATING AND AMPLIFYING SPECIFIC DNA FRAGMENTS: how can a specific segment of DNA be isolated from an entire genome? How can it be isolated in quantities sufficient to analyse features of DNA i.e sequence and protein product?

Answer 27

Researchers can create large samples of DNA by tricking replication machinery: - within live bacterial cells (in vivo) - donor DNA fragments are inserted into specially designed plasmid that will "carry" and amplify gene of interest - vectors - OR in a tube (in viTro) - Polymerase Chain reaction (PCR)

Question 28

In VIVO vs VITRO TECHNIQUES AND SIMILARITIES?

Answer 28

in vivo = tricking replication machinery of bacterium into amplifying recombinant DNA containing the gene in vitro = in test tube using PCR. BOTH METHODS use basic principles of molecular biology: - ability of specific proteins to binds DNA - ability of complementary single-stranded nucleic acid segments to hybridise together (primer)

Question 29

MOLECULAR TECHNIQUES ARE USED TO ISOLATE, RECOMBINE AND AMPLIFY GENES: explain the steps

Answer 29

1. Isolate DNA segement or gene from remainder of DNA, the to make copies of it for further analysis

Question 30

A key event in 1960s was identification of restriction enzymes (REs); what are they, why useful? which one is he most useful?

Answer 30

REs = recognise, make double-stranded breaks in DNA at specific nucleotide sequences Produced NATURALLY by BACTERIA as DEFENCE against VIRUSES. Protects its own DNA by METHYLATION. Many types of REs - TYPE 2 most useful as cut DNA at defined sites that are PALINDROMIC. Some make STAGGERED CUTS (Cohesive ends), other CUT STRANDS STRAIGHT ACROSS (blunt ends)

Question 31

Understanding Restriction enzymes...what , how cut, recognition

Answer 31

1. Some restriction enzymes, such as HINDIII, make staggered cuts in DNA,... 2. ...producing single stranded, COHESIVE (sticky) ends. 3. Other restriction enzymes, such as PvuII,... 4. ...cut both strands of DNA straight across creating BLUNT ends 5. MOST ENZYMES RECOGNISE SEQUENCE OF 4 OR 6 bp. 6. MOST RECOGNITION SEQUENCES ARE PALINDROMIC (sequences read same forward and back) 7. DNA molecules cut with the same restriction enzyme have complementary sticky ends that pair if fragments are mixed together . 8. The nicks in the sugar-phosphate backbone of the 2 fragments can be sealed by DNA ligase. 9. Cohesive or sticky ends - complementary to each other. 10. Spontaneously pair to connect fragments. 11. Can be joined by DNA Ligase, which seals nicks between sugar-phosphate groups.