Unit 12: Molecular biology techniques

Question 1

define locus

Answer 1

specific site of a gene on a chromosome

Question 2

define allele

Answer 2

one of two or more alternative forms of a gene

Question 3

Define polymorphism

Answer 3

Presence of more than one allele of a gene/locus in a population at a frequency greater than that of new arising mutation

Question 4

genotype vs. phenotype

Answer 4

genotype: entire genetic makeup
phenotype: physical and physiological characteristics. the specific trait associated with a particular allele

Question 5

describe dominant mutations

Answer 5

• when the mutant phenotype is expressed with the WT allele present
• typically gain of function that results in increased activity and new function
• there is sometimes loss of function

Question 6

name and describe types of loss of function in dominant mutations

Answer 6

haploinsufficiency: one gene copy is inactive, the functional copy does not provide enough gene product for the WT phenotype

dominant-negative: mutation interferes with WT protein function

Question 7

describe recessive mutations

Answer 7

• WT phenotype expressed with WT allele present
• typically gene is inactivated partially or completely resulting in loss of function

Question 8

Loss of function mutations vs. conditional loss-of-function mutation

Answer 8

• these are null mutations
• loss of function mutations include point mutations and deletions. these completely get rid of gene function
• conditional loss-of-function mutations depend on temperature. Usually dysfunctional at high temperatures

Question 9

Describe conditional mutations

Answer 9

• mutations cause proteins to be functional only under certain conditions such as temperature
• permissive temperatures produce the WT phenotype
• non permissive temperatures produce the mutant phenotype
• this helps us study lethal mutations

Question 10

what is the purpose of genetic complementation experiments?

Answer 10

genetic complementation- growth indicates different loci b/c of normal function
non-complementation- no growth indicates same loci but different alleles

Question 11

what is the purpose of epistasis experiments?

Answer 11

experiments help determine order of function of gene products in a pathway

Question 12

explain how gene loci are mapped in the genome

Answer 12

• gene loci are mapped by determining distance to known loci

Question 13

what is complementation

Answer 13

• when several genes contribute to phenotype
• crossing heterozygotes with mutant recessive alleles in different loci will restore WT phenotype

Question 14

what is epistasis

Answer 14

one gene’s alleles mask the effect of another gene’s alleles

Question 15

what are single nucleotide polymorphisms (SNP)

Answer 15

single base sequence variation between individuals at a particular point in the genome

Question 16

what are the required components and general process of DNA cloning

Answer 16

1. generate recombinant vector
2. transfer recombinant vector into host cell
3. produce multiple copies of recombinant DNA by host
4. select/identify recombinant host cells

Question 17

how do you generate a recombinant vector

Answer 17

• cut source DNA at edges of gene
• select a suitable carrier DNA (vector) that is capable of self replication in a host
• insert gene covalently into vector

Question 18

explain the principle of electrophoresis and how it is applied to DNA

Answer 18

separate based on mass using electric field
- it is used for
1. qualitative DNA analysis (determine purity of plasmid prep)
2. DNA purification: isolation of restriction fragments from gel
3. DNA-protein interaction studies: electrophoretic mobility shift assay

Question 19

what do restriction enzymes do?

Answer 19

catalyze single strand breaks (nicks) on either DNA strand or double-strand breaks in DNA
- they hydrolyze phosphodiester bonds of DNA. producing free 3’-OH group and 5’-phosphate group

Question 20

how is host DNA protected from restriction enzymes

Answer 20

methylation

Question 21

where do restriction enzymes cleave on foreign DNA

Answer 21

restriction enzyme cleaves unmethylated foreign DNA within 4-8 base pair recognition site
- they recognize inverted repeats in DNA (aka palindromes) and cleave at the 3’ or 5’ site of/or at symmetry axis

Question 22

are restriction enzymes used to cut out DNA segments of interest?

Answer 22

yes

Question 23

define palindrome

Answer 23

• inverted sequence repeats that read in the same direction on each strand

Question 24

what does choosing a cloning vector depend on?

Answer 24

1. size of DNA insert
2. efficiency of the host cell to uptake vector
3. amplification

Question 25

the three essential properties cloning vectors must possess

Answer 25

1. restriction site that allows insertion of target DNA
2. autonomous replication in a host cell so that amplification of target DNA can occur independent of host DNA synthesis
3. a selectable feature that allows transfected host cells containing the vector to be identified from other cells w/o vector

Question 26

describe plasmid vectors

Answer 26

• circular molecules
• replicate autonomously
• transfer to host via transformation
• selective marker is antibiotic resistance gene(s)
• can accommodate up to 15 kb

Question 27

describe bacteriophages

Answer 27

• viruses infecting bacteria (e.g. lambda)
• transfer to host via transduction (aka phage infection)
• can insert up to 15-25 kb

Question 28

describe cosmids

Answer 28

• plasmids with lambda segments or lambda phages
• accommodate larger inserts
• can hold up 20 kb

Question 29

describe bacterial artificial chromosomes (bac)

Answer 29

• plasmids for very large fragments
• can hold 150-350 kb
• maintained at low copy numbers due to par genes regulating distribution of plasmids at division
• includes selectable markers and genes that regulate plasmid distribution

Question 30

Yeast artificial chromosomes (YAC)

Answer 30

• contain all elements required for maintenance in eukaryotic cells ( origin, centromere, telomeres) and selective markers
• Circular and linear form
  circular for rapid replication and stable storage
  removable product that linearizes for transformation and restriction site for cloning
• can insert 150-3000 kb

Question 31

what are DNA libraries

Answer 31

collection of DNA molecules stored in vectors

Question 32

genomic libraries vs. cDNA libraries

Answer 32

Genomic libraries:
- store complete genome of an organism
- tissue- independent
- contain complete coding region, including introns
- obtained from restriction enzyme digestion of the entire genome of an organism
- vectors used: plasmids, cosmid, lambda phage, BAC, YAC
- large fragments

cDNA (complementary DNA) libraries:
- store mRNA converted to DNA of a cell/tissue.
i.e all expressed genes of particular cell/tissue
- obtained from the mixture of mRNAs expressed by a particular tissue under given physiologic conditions
- clones lacks introns and a promoter region
enzymes: spliceosome, reverse transcriptase, and DNA polymerase
- vectors used: plasmid, lambda phage,
- small fragments

Question 33

methods that use nucleic acid hybridization

Answer 33

• Gene analysis- based on reversible denaturation of DNA and complementarity
• southern blotting
• northern blotting
• western blotting
• eastern blotting
• south-western blot

Question 34

what does gene analysis by hybridization utilize

Answer 34

probes- short ssDNA or RNA molecules complementary to specific region. are meant to hybridize with target DNA
- they are labeled for detection using radioactive isotopes, fluorescent/biotin tags
- their design is based on sequence formation

Question 35

describe southern blotting

Answer 35

• DNA is denatured during transfer onto filter membrane. this allows probes to access.
• uses labeled DNA probes
• filter hybridized with probe to detect specific DNA fragment
• detects presence or absence of genes or alleles
• detects specific restriction fragment out of the millions present in a restriction digest

Question 36

describe northern blotting

Answer 36

• detects RNA with labeled DNA probes
• useful in determining whether a specific mRNA is expressed in a particular tissue

Question 37

describe western blotting

Answer 37

• detects proteins by using labeled antibodies

Question 38

describe south-western blotting

Answer 38

-detects DNA-binding proteins with labeled DNA probes
- useful for detecting expression of transcription factors that interact with regulatory sequences controlling specific genes

Question 39

describe eastern blot

Answer 39

• detects posttranslational modifications

Question 40

what does PCR do?

Answer 40

amplifies specific regions of DNA in vitro replication using DNA polymerase
-oligonucleotides act as replication primers
- forward and reverse primers selected to flank target region
- does not require cloning

Question 41

steps of PCR

Answer 41

1. denature template using heat
2. lower temp to allow hybridization of primers
3. extend strand using heat-stable DNA polymerase
4. repeat multiple cycles (25-45)
   heat stable taq polymerase

Question 42

what are PCR applications

Answer 42

1. exponential amplification of DNA: increases efficiency in recombinant DNA procedures
2. isolation and subcloning of specific segment of genomic DNA
   - design of primers with restriction site to facilitate sub cloning
3. preparation of DNA probes
4. diagnostics
5. quantification of DNA or cDNA by qPCR (quantitative/real time PCR)
6. forensic DNA analysis
7. Gene expression analysis by Reverse transcriptase PCR

Question 43

what is the Sanger dideoxy method

Answer 43

• in vitro synthesis of one strand (template based)
• uses didoexynucleotides to interrupt DNA synthesis using the unknown DNA as the template
• length is determined by the last base in sequence

Question 44

describe Sanger dye termination sequencing

Answer 44

• daughter strand is denatured and separated by electrophoresis
• capillary gel electrophoresis separation for high resolution
• automated process using sequnators

Question 45

how is the efficiency of net-generation sequencing achieved?

Answer 45

through pre-amplification of templates by PCR

Question 46

how are genome sequences accessible

Answer 46

through databases:
- Genbank
- EMBL

Question 47

How are sequences compared

Answer 47

using BLAST (Basic local alignment search tool)
- nucleic acid or protein sequences
- uses similarity scores (p-values)

Question 48

ways of whole genome sequencing

Answer 48

early on: physical mapping and ordered sequencing of restriction fragments

shotgun approach: random sequencing and in silico assembly of single contiguous assemblies

Question 49

gene expression studies

Answer 49

1. northern blotting- mRNA expression. through electrophoresis separation, denaturation and blot transfer, hybridization w/ mRNA specific probe for mRNA detection
   - semi-quantitative comparison w/ standard mRNA signal
2. in situ hybridization- detection of mRNA using labeled DNA or RNA probes. analysis of spatial and/or temporal expression patterns
3. DNA microarrays- uses high density DNA chips to screen expression of large # of genes
   - isolates total mRNA, requires RT, and flourescently labeled dye & hybridization
   - allows analysis of comparative expression in clinical and lab settings

Question 50

gene functional studies

Answer 50

• in vivo gene expression
• genetically modified animals

Question 51

novel methods for gene expression analysis

Answer 51

4. RNAseq- applying sequencing methods to RNA for transcriptome analysis. does not require prior knowledge of RNA sequences
5. ribosome profiling- identify mRNA being actively translated
   -isolated RNA and RNAse digest of free RNA followed by sequencing
   -isolation and sequencing of remaining RNA
   - requires reverse transcription of RNA sequences into cDNA

Question 52

strategies to manipulate gene expression

Answer 52

1. in vivo gene expression- transfection of foreign DNA into eukaryotic cells
   - constitutive (continuous) or inducible (conditional) expression
   - untagged or tagged target protein
   - types of transfection: transient- expression is from cDNA in plasmid DNA; Stable (transformation)- expression from cDNA is integrated into host chromosome
2. genetically modified animals-
   transgenic animals: animals carry foreign genes into genome and pass to offspring.
   -by injecting trans gene into fertilized egg, random insertion by NHEJ, Selection and breed offspring
   knockout animals: targeted gene inactivation by HR (deleted gene)
   knockin animals: targeted gene replacement (modified gene)
3. IoxP Cre recombination system- for tissue specific targeted gene disruption by somatic cell recombination.
   -viral recombinase Cre recognizes specific IoxP sequences. cleaves intervening sequences between IoxP sites.
   - targeted gene knockout
4. CRISPR- bacterial defense using non-coding RNA
   - targets bacteriophages.
   integrates phage DNA fragment into specific genomic regions
   when reinfected, sequences cleaves due to complementarity w/non-coding RNA